Adult Stem Cells/Progenitor Cells and Stem Cell Proteins for Treatment of Eye Injuries and Diseases

ABSTRACT

The present invention encompasses methods and compositions for treating an ocular disease, disorder or condition in a mammal. The invention includes a population of mesenchymal stromal cells that possess anti-inflammatory, anti-apoptotic, immune modulatory and anti-tumorigenic properties. The invention includes administration of TSG-6, STC-1, or a combination thereof to the ocular as a treatment for an ocular disease, disorder or condition in a mammal.

This application is a divisional of application Ser. No. 13/643,592,filed Oct. 26, 2012, which is the national phase application of PCTApplication No. PCT/US2011/000771, filed May 3, 2011, which claimspriority based on provisional application Ser. No. 61/464,172, filedFeb. 28, 2011, and provisional application Ser. No. 61/330,735, filedMay 3, 2010, the contents of which are incorporated by reference intheir entireties.

This invention was made, in part, using funds obtained from the U.S.Government (National Institutes of Health Grant No. R21EY020962), andthe U.S. Government therefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Stem cells have significant therapeutic potential. In a non-limitingaspect, the present invention is directed to using mesenchymal stemcells to treat eye injuries and diseases. Other non-limiting aspects aredirected to the therapeutic use of mesenchymal stem cells for treatingdiseases which include those of the lung and heart. Another non-limitingaspect of the present invention is directed to the use of anti-apoptoticand anti-inflammatory proteins such as STC-1 and TSG-6, which areexpressed by mesenchymal stem cells, for treating the above-mentioneddiseases and disorders.

The cornea, apart from being an important component of the refractivesystem of the eye, serves as a biologic and physical barrier byprotecting the interior structures of the eye from environmentalinsults. The transparency of the cornea and, consequently, visual acuityare both dependent upon the integrity and functionality of the outermostlayer, the epithelium. Most of the diseases occurring in cornealepithelium (i.e., corneal surface diseases) are accompanied by sterileinflammation and defects in wound healing. Therapies for these diseasesremain problematic.

The most severe form of corneal surface diseases is limbal epithelialstem cell deficiency (LSCD). It can be primary as a result of inheritedeye disease, but more commonly it is the result of acquired conditionssuch as chemical or thermal burn injuries, systemic autoimmune disease,contact lens keratopathy, recurrent ocular surgeries, or Stevens-JohnsonSyndrome (SJS). The incidence of chemical burns is 500,000 cases peryear and accounts for 7 to 18% of the 2.5 million cases of ocular traumaseen in emergency departments each year in the U.S. (Melsaether et al.,2009). The annual incidence of SJS that causes severe LSCD is 2.6 to 7.1cases per 1 million people or about 200,000 people in the U.S.population (Foster et al., 2008). LSCD not only inflicts enormouspsychological stress on patients, but it also carries an economic burdenin terms of loss of productivity and prohibitive health care costs overthe lifetime of the patients.

The current treatments of LSCD include anti-inflammatory drug therapy(e.g. steroids) in the early phase and transplantation of limbalepithelial stem cells (LESCs) in the late stage (Limb and Daniels,2008). However, the anti-inflammatory drugs currently available aredisappointing and are unable to prevent subsequent loss of LESCS. Thecurrent strategies for correcting LSCI by transplanting LESCs from apatient's healthy eye (limbal autograft), a living-relative, orcadaveric donors (limbal allograft) have many drawbacks (Ilari et al.2002; Solomon et al, 2002; Cauchi et al. 2008). The limbal autograftcarries the risk of creating LSCD in the donor eye (Jenkins et al.,1993) and cannot be used in patients with bilateral LSCD. Limbalallografts require long-term systemic immunosuppression because of thepresence of HLA-DR antigens and Langerhans cells in the graft. Even withimmunosuppression, immunological rejection occurs in 42.9% to 64.0%patients after limbal allografts (Rao et al., 1999; Tseng et al., 1998;Shi et al., 2008; Reinhard et al., 2004; Tsubota et al., 1999).Currently, ex vivo cultivation and transplantation of LESCs are beingused for LSCD in some centers (summarized in Shortt et al., 2007).However, the putative stem cells observed in the limbus have not yetbeen reproducibly isolated or fully characterized. Moreover, the mainclinical limitation for using cultured LESCs therapeutically is theavailability of an autologous donor tissue. Although cultured allogeneiccells may be used in combination with systemic immunosuppression, donorallogeneic LESCs do not survive beyond a period of 9 months (Daya etal., 2005; Sharpe et al., 2007). Effective suppression of inflammationat the ocular surfaces is critical for success. Therefore, the therapiesthat are the goal of the present application can provide an importantadvance to the current therapy.

In addition to vision-threatening LSCD, there are a number of ocularsurface diseases accompanied by corneal inflammation and defects inwound healing, such as keratoconjunctivitis sicca, recurrent cornealerosion, or post-refractive surgery keratitis. The most common is dryeye syndrome that affects nearly 10% of the U.S. population (Moss etal., 2000; Sehaumberg et al., 2003; Moss et al., 2008). As many as 20 to30 million people in the United States have exhibited early signs orsymptoms of dry eye, and an estimated 6 million women and 3 million mensuffer from advanced effects of dry eye that alter the quality of life(Mertzanis et al., 2005; Miljanovic et al., 2007). Unfortunately, mostof the currently-available therapeutic agents are only palliative andnone of them abolish signs and symptoms of dry eye completely(Pflugfelder, 2007). Several studies (Clegg et al., 2006; Reddy et al.,2004; Callaghan et al., 2007) have established that the economic impactof dry eye syndrome is also substantial, in terms of both direct medicalcosts (e.g. for medications and physician visits) and indirect costs(e.g. lost work time and impaired productivity). In effect, there are noefficient and safe therapeutic strategies for diseases of the cornealsurface.

Retinal degenerations, including age-related macular degeneration (AMD)and retinitis pigmentosa (RP), are the leading causes of legal blindnessin the United States. AMD and RP share clinical and pathologic featuresincluding end-stage blindness due to photoreceptor and/or retinalpigment epithelium (RPE) cell death. Of special importance is thatapoptosis of photoreceptors has been shown to be a prominent feature ofhuman AMD (Dunaief, et al., Arch. Ophthalmol., Vol. 120, No. 11, pgs.1435-1442 (2002); Xu, et al., Trans. Am. Ophthalmol. Soc., Vol. 94, pgs.411-430 (1996)). and RP (Cottet, et al., Curr. Mol. Med., Vol. 9, No. 3,pgs. 375-383 (2009); Doonan, et al., Curr. Neurosci. Res., Vol 1, No. 1,pgs. 47-53 (2004)) and is the underlying feature in many of the animalmodels of retinal degeneration (Doonan, 2004; Yu, Invest. Ophthalmol.Vis. Sci., Vol. 45, No. 6, pgs. 2013-2019 (2004); Katai, et al., Invest.Ophthalmol. Vis. Sci., Vol. 40, No. 8, pgs. 1802-1807 (1999); Katai, etal., Jpn. J. Ophthalmol., Vol 50, No. 2, pgs. 121-127 (2006)). Reactiveoxygen species (ROS) have been implicated in the initiation and/orexacerbation of cell death in AMD (Fletcher, et al., Ophthalmic Res.,Vol. 44, No. 3, pgs. 191-198 (2010); Beatty, et al., Surv. Ophthalmol.,Vol. 45, No. 2, pgs. 115-134 (2000); Winkler, Mol. Vis., Vol. 5, pg. 32(1999); Johnson, Curr. Opin. Cln. Nutr. Metab. Care, Vol. 13, pgs. 28-33(2010); Totan, et al., Curr. Eye Res., Vol. 34, No. 12, pgs. 1089-1093(2009)) and antioxidant vitamin therapy is currently one of themainstays of treatment in non-exudative AMD and RP (Johnson, 2010;Hartong, et al., Lancet, Vol. 368, pgs. 1795-1809 (2006)). Although notcurative, reduction of risk of disease and stabilization of vision havebeen observed following antioxidant vitamin therapy (Flectcher, 2010;Beatty, 2000; Johnson, 2010; Hartong, 2006). Moreover, two of the topmodifiable risk factors in AMD-smoking and light exposure—are thought toinjure photoreceptors or RPE through ROS-mediated damage (Flectcher,2010; Johnson, 2010). In addition, oxidative damage studies in animalmodels of RP have implicated ROS as partially responsible for theapoptotic loss of photoreceptors (Komeima, et al., Proc. Nat. Acad.Sci., Vol. 103, No. 30, pgs. 11300-11305 (2006); Shen, et al., J. Cell.Physiol., Vol. 203, No. 3, pgs. 457-464 (2005)). Further evidenceincludes studies demonstrating that antioxidant based therapies slowphotoreceptor death in animal models of RP (Komeima, et al; J. Cell.Physiol., Vol. 213, No. 3, pgs. 809-815 (2007); Galbinar, et al., J.Ocul. Pharmacol. Ther., Vol 25, No. 6, pgs. 475-482 (2009); Chen, etal., Nat. Nanotechnol., Vol. 1, No. 2, pgs. 142-150 (2006)) and decreaseprimary retinal cell (Chen, 2006; Chucair, et al., Invest. Ophthalmol.Vis. Sci., Vol. 48, No. 11, pgs. 5168-5177 (2007)) or RPE cell death invitro (Cao, et al., Exp. Eye Res., Vol. 91, No. 1, pgs. 15-25 (2010);Kim, et al., Invest. Ophthalmol. Vis. Sci., Vol. 51, No. 1, pgs. 561-566(2010)). Photoreceptors are sensitive particularly to oxidative stressfor several reasons: (a) oxygen consumption by the retina is greaterthan any other tissue (Beatty, 2000); (b) photoreceptor outer segmentsreside near a rich vascular supply and contain high concentrations ofpolyunsaturated fatty acids (PUFAs) that undergo oxidation when oxygenis present (Winkler, 1999); (c) lipofuscin accumulates in the RPE duringaging and is thought to be a source of ROS that is toxic to the RPE(Beatty, 2000); and (d) photoreceptors are subject to repeatedphotochemical injury, a process known to produce ROS (Beatty, 2000).These findings in conjunction with our preliminary data provide a strongrationale to test new stem cell based therapies known to reduceROS-mediated apoptosis in currently incurable degenerative diseases ofthe retina.

Inflammation is being shown increasingly to play a role in a number ofdiseases, such as in myocardial infarction, stroke, Alzheimer's diseaseand atherosclerosis, for example. Thus, the present invention in anon-limiting aspect, is directed to the use of mesenchymal stem cellsand the role of certain proteins, such as TSG-6, in treating myocardialinfarction and lung diseases.

In summary, there are no effective drugs to treat corneal inflammationand ulceration caused by chemical injury to the cornea. In addition,there is a need for additional therapies for macular degeneration. Therealso are needs for additional therapies for diseases of the lung andheart. Thus, the present invention is directed to the use of mesenchymalstem cells and stem cell proteins as therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

FIG. 1, comprising FIGS. 1A through 1C, is a series of images depictinginduction of corneal inflammation and neovascularization, one weekpost-injury. FIG. 1A is an image demonstrating that on slit lampexamination, corneal inflammation with active new vessels was observed,FIG. 1B is an image of immunofluorescent staining for VEGF showing themarked increase in VEGF expression in cornea. VEGF as green and thenuclei counterstained as blue. FIG. 1C is an image depictingHematoxylin-eosin staining revealing that the cornea was denselyinfiltrated with inflammatory cells.

FIG. 2, comprising FIGS. 2A and 2B, is a series of images depictingapplication of cells to cornea. FIG. 2A is an image of a 6-mm-diameterhollow plastic tube emplaced to keep the eye open and the cells or mediaapplied to the cornea into a customized applicator. FIG. 2B is an imagedepicting engraftment of MSCs in the cornea confirming by identificationof PKH26-labeled cells in corneas by fluorescein microscopy.

FIG. 3, comprising FIGS. 3A through 3L, is a series of images depictingphotography of cornea one (FIGS. 3A-3D), two (FIGS. 3E-3H), and threeweeks (FIGS. 3I-3L) post-injury. With time, neovascularization andopacity markedly decreased in the corneas with MSCs (FIGS. 3D, 3H, 3L)or MSC-CM media three times (FIGS. 3C, 3G, 3K), while increased in thecontrol (FIGS. 3A, 3E, 3I). Corneas treated with MSC-CM once (FIGS. 3B,3F, 3J) showed the intermediate outcome.

FIG. 4, comprising FIGS. 4A through 4D, is a series of images depictingHematoxylin-eosin staining of cornea three weeks post-injury. Control(FIG. 4A) and corneas treated with MSC-conditioned media once (FIG. 4B)were densely infiltrated with inflammatory cells in the stroma andgoblet cells in the epithelium. The infiltration was markedly reduced inthe corneas with MSC-conditioned media three times (FIG. 4C) or MSCs(FIG. 4D).

FIG. 5, comprising FIGS. 5A through 5D, is a series of imagesdemonstrating inflammation-related cytokine expression evaluated byELISA. IL-2 and IFN-γ were repressed in the corneas treated with MSCs orMSCs-conditioned media three times (MSC-CM II) compared to the control.

FIG. 6, comprising FIGS. 6A through 6D, is a series of images depictingreal-time PCR for angiogenesis-related cytokines. Upregulation of TSP-1was observed in the corneas treated with MSC or MSC-conditioned mediathree times (MSCCM II), compared to the control. MMP-2 and MMP-9 weredownregulated in the MSC group. There were no differences in theexpression of VEGF. Values were expressed as folds relative to freshcorneas without an injury.

FIG. 7 is an image depicting cytotoxicity test of human cornealepithelial cells (HLECs) after chemical damage. When cultured withhMSCs-derived medium for 48 hours, damaged HLECs were significantlydecreased compared to HLECs without hMSCs-conditioned medium.

FIG. 8 is an image depicting cytokine secretion evaluated by ELISA. Theexpression of VEGF, MMP-9, MMP-2, and TSP-I were quantified in variouscocultures of hMSCs/hCECs/hPBMCs. The hCECs were prepared aftertreatment with 15% ethanol for 30 sec. Data represent at least threeexperiments.

FIG. 9, comprising FIGS. 9A through 9F, is a series of imaged depictingassays for fate of hMSCs infused into mice. FIG. 9A is an imagedepicting clearance of human Alu sequences from blood after IV infusionof about 2×10⁶ hMSCs into mice. Values are mean+/−_(−/−) SD; n=6. FIG.9B is an image depicting standard curves for real time PCR assays ofhuman Alu sequences in 7 organs. Values indicate ΔΔCt for primers formouse/human GAPDH genes and Alu sequences on same samples. FIG. 9C is animage depicting tissue distribution of human Alu sequences 15 mm afterIV infusion of about 2×10⁶ hMSCs into mice. Values are mean+/−SD; n=6.FIG. 9D is an image depicting standard curves for real time RT-PCRassays of human mRNA for GAPDH. Values indicate AACt for primers formouse/human GAPDH genes and cDNA for human-specific GAPDH on samesamples. FIG. 9E is an image depicting kinetics of hMSCs in lung and 6other tissues after IV infusion of about 2×10⁶ hMSCs, Values aremean+/−SD; n=6. FIG. 9F is an image depicting appearance of hMSCs inheart after IV infusion of about 1×106 hMSCs I day after permanentligation of the left anterior descending coronary artery.

FIG. 10, comprising FIGS. 10A through 10F, is a series of imagesdepicting activation of hMSCs to Express TSG-6. FIG. 10A is an imagedepicting real-time RT-PCR for human-specific mRNA in lung 10 hr afterIV infusion of 2×10⁶ hMSCs. Values are fold increase over values forcultured hMSCs, normalized by ΔΔCt for hGAPDH. Symbols: hMSCs con,sample of hMSCs added to lung from control mouse before extraction ofRNA; hMSCs IV 1 and 2, samples from lungs of 2 mice 10 hr after IVinfusion of hMSCs. FIG. 10B is an image depicting real-time RT-PCR forhuman TSG-6 in mouse lung. About 2×10⁶ hMSCs were infused IV into naïvemice (IV-nor) or mice at 1 hr after Ml (IV-MI) and lungs were recovered0.25 hr to 24 hr later. Values are +/−SD; n=2 or 3 for normal mice; n=6for MI mice. FIG. 10C is an image depicting real-time RT-PCR for TSG-6in hMSCs and human fibroblasts from the same donor incubated inserum-free medium with 10 ng/ml TNF-α for 24 or 48 hr. Results with twopassages of the same cells are shown. Values are +/−SD; n=3. FIG. 10D isan image depicting ELISAs for TSG-6 in medium from hMSCs and humanfibroblasts incubated in serum-free medium with 10 ng/ml TNF-α for 48hr. Values are ±1=SD; n=3. FIG. 10E is an image depicting real-timeRT-PCR assays for TSG-6 of control hMSCs (Con), hMSCs treated withtransfection reagents only (no siRNA), hMSCs transfected with ascrambled siRNA (scr siRNA) or hMSCs transduced with TSG-6 siRNA (TSG-6siRNA). Cells were incubated with or without 10 ng/ml TNF-α for 6 hr.Values are +/−SD; n=3. FIG. 10F is an image depicting ELISAs for TSG-6in medium after incubation of cells with or without TNF-α for 48 hr.Symbols: as in FIG. 10E. Values are +/−SD; n=3.

FIG. 11, comprising FIGS. 11A through 11E, is a series of imagesdepicting assays of serum and heart. FIG. 11A is an image depicting anassay for cardiac troponin I in serum 48 hr after MI. Values are +/−SD;p<0.01 with n=3 (Normal) or 6 mice (MI) per group. FIG. 11B, is an imagedepicting plasmin activity in serum 48 hr after MI. Symbols: Normal,naïve mice; MI only; hMSCs, 2×10⁶ hMSCs infused IV 1 hr after MI; scrsiRNA, 2×10⁶ hMSCs transduced with scrambled siRNA infused IV 1 hr afterMI; TSG-6 siRNA, 2×10⁶ hMSCs transduced with TSG-6 siRNA infused IV 1 hrafter MI; rhTSG-6, 30 μg rhTSG-6 protein infused IV 1 hr and again 24 hrafter MI. Values are _(+/−)SD; p<0.01 with n=3 mice per group. N.S.=notsignificant. FIG. 11C, is an image depicting hearts assayed for pro- andactive-matrix MMP9 on a gelatin zymogen gel 48 hr after MI. Image isreversed. Symbols: as in FIG. 11B. FIGS. 11D and 11E are imagesdepicting granulocyte and monocyte infiltration in the heart 48 hr afterMI. Sections stained with anti-Ly-6G and Ly-6C.Symbols: as in FIG. 11Bexcept 100 μg rhTSG-6 protein was infused IV 1 hr and again 24 hr afterMI. Magnification ×4. Scale bars, 250 μm. Values are +/−SD; n=3 or 4 foreach group. ** p<0.001; N.S.=not significant.

FIG. 12, comprising FIGS. 12A through 12F, is a series of imagesdepicting assays of Infarct Size 3 wk after MI. Each heart was cut fromthe apex through the base into over 400 sequential 5 μm sections andstained with Masson Trichrome. Every 20th section is shown from typicalspecimens. FIG. 12A is an image depicting MI. Heart with no treatment.FIG. 12B is an image depicting MI+hMSCs. 2×106 hMSCs infused IV 1 hrafter MI. FIG. 12C is an image depicting MI+scr siRNA, 2×106 hMSCstransduced with scrambled siRNA infused IV 1 hr after MI. FIG. 12D is animage depicting MI+TSG-6 siRNA. 2×106 hMSCs transduced with TSG-6 siRNAinfused IV 1 hr after MI. FIG. 12E is an image depicting MI+hTSG-6 100μg rhTSG-6 protein infused IV 1 hr and again 24 hr after MI. FIG. 12F isan image depicting Infarct size measurements (%) obtained by midlinelength measurement from every 10^(th) section of the infarct area for atotal of 20 sections per heart (Takagawa et al., 2007). Values are+/−SD; n−3 or 4 mice per group; ***p<0.0001 compared to Ml controls;N.S. not significant compared to MI controls; *p<0.05 for MI+MSCs versusMI+rhTSG-6.

FIG. 13 is an image demonstrating that STC-1 was required and sufficientfor reduction of apoptosis of lung epithelial cell line made apoptoticby incubation at low pH in hypoxia. Upper left and right: Cultures ofA549 cells became apoptotic when incubated for 24 hours in 1% oxygen atpH 5.8 or 5.5. However, coculture of A549 cells in transwells with MSCsreduced the apoptosis. Lower left: Apoptosis of A549 cells was inhibitedby rhSTC-1, and the effects were reversed by anti-STC-1 antibodies.Lower right: MSCs transduced with siRNA for STC-1 were less effectivethan control MSCs in decreasing apoptosis of A549 cells in the transwellexperiment.

FIG. 14 is a schematic of a strategy to search for additional, noveltherapeutic factors produced by hMSCs in response to corneal injury.

FIG. 15 is a series of imaged demonstrating that conditioned medium frompre-activated MSCs and rhSTC-1 had the greatest effects in hMSCsimproving the viability, increasing the proliferation, and inhibitingthe apoptosis of damaged hCEPs.

FIG. 16, comprising FIGS. 16A through 16F, is a series of imagesdemonstrating that intracameral injection of TSG-6 (2 ug) decreasedcorneal opacity and neovascularization in cornea after injury. FIGS.16A-16F are photo images of cornea. FIGS. 16A-16C are images depictingPBS-treated control. FIGS. 16D-16F are images depicting TSG-6-treatedcornea.

FIGS. 16A, 16D depict postoperative day 3. FIGS. 16B, 16E depictpostoperative day 7. FIGS. 16C, 16F depict postoperative day 21. Bottomframes: Clinical evaluations of opacity (left frame) andneovascularization (right frame) of the cornea.

FIG. 17, comprising FIGS. 17A through 17G, is a series of imagesdemonstrating intracameral injection of TSG-6 (2 ug) decreased theinfiltration of neutrophils and production of MMP-9 in cornea afterinjury. (FIGS. 17A-17D) Hematoxylin-eosin staining of cornea. (FIGS.17A, 17B) PBS-treated cornea. (FIGS. 17C, 17D) TSG-6-treated cornea.(FIGS. 17A, 17C) Postoperative day 3. (FIGS. 17B, 17D) Postoperative day21. (FIG. 17E) Myeloperoxidase assay. (FIG. 17F) Gel zymography forMMP-9. (FIG. 17G) ELISA for total and active MMP-9.

FIG. 18 is a graph depicting correlation between clinical opacity andMPO amount in cornea at post-injury 3 days.

FIG. 19, comprising FIGS. 19A through 19D, is a series of imagesdemonstrating that TSG-6 up to the concentration of 2 ug is effective inreducing corneal opacity, inflammation, and MMP-9 production. (FIGS.19A-19D) Photography of cornea. (FIG. 19A) PBS-treated cornea. (FIG.19B) TSG-6 0.02 ug-treated cornea. (FIG. 19C) TSG-6 0.2 ug-treatedcornea. (FIG. 19D) TSG-6 2 ug-treated cornea. (FIG. 19E) Myeloperoxidaseassay and clinical grading of opacity. (FIG. 19F) Gel zymography andELISA for MMP-9.

FIG. 20 is an image depicting real time PCR for inflammatory cytokinesand chemokines. RQ: relative gene expression. Comparisons are betweenvehicle treatment (PBS) and TSG-6 treatment (2 micrograms) onpost-operative days (POD) 3, 7, and 21.

FIG. 21 is an image depicting ELISA for cytokines and chemokines.Conditions as in FIG. 20.

FIG. 22 is an image demonstrating that TSG-6 delayed the timeneutrophils started to infiltrate and arrived at its peak as well asdecreased the amount of infiltrated neutrophils. The expression patternof chemokines and cytokines showed similar kinetics. Lower two frames:Blood levels of MPO and leukocytes (WBC).

FIG. 23. Two injections of STC-1 rescued retinal degeneration in therhodopsin mutant transgenic rat. Upper frame: representative posteriorsegment histology showed thickened ONL in STC-1 treated eyes compared toUI controls. The ONL is the outer nuclear layer of the retina thatcontains the nuclei of the rods and cones. Lower frame: representativeplot of ONL layer thickness taken from a total of 54 measurements (27superior retina and 27 inferior retina) demonstrated STC-1 significantlyimproved ONL thickness compared to UI controls.

FIG. 24. Age related loss of mRNAs for photoreceptors in RCS rat.qRT-PCR analysis for the photoreceptor genes: rhodopsin, phosducin,neural retina leucine zipper, and recoverin. Expression of these genesdecreases over time in the RCS rat. For qPCR methods see Lee, 2008.

FIG. 25. Rescue of mRNAs for photoreceptors by intravitreal injection ofSTC-1 in RCS rat. qRT-PCR analysis for photoreceptor genes was conductedas described with respect to FIG. 24.

FIG. 26 MSCs survive in the vitreous cavity following injection. LeftPanel: standard curve with human specific qRT-PCR for human GAPDH mRNAas a reflection of viable MSCs (see Lee, 2009 for Methods). Varyingnumbers of human MSCs added to whole globe just before RNA wasextracted. Right Panel: Recovery of viable human cells 4 days afterintravitreal injection of 100,000 human MSCs.

FIG. 27. Activation of expression of STC-1 by culture of human MSCs inhanging drops so that the cells coalesced into spheroids. High densitymonolayer (Adh High), spheroids (Sph 25k), and spheroid derived MSCs(Sph 25k DC) were transferred to 6 well plates containing 1.5 mlcomplete culture medium (CCM) and either 200,000 MCSs from high densitycultures, eight 25k spheroids, or 200,000 MSCs. After 24 hours, mediumwas recovered for ELISAs and cells were lysed for protein assays. Figureadapted from (Bartosh, 2010). The results demonstrated an over 20-foldincrease in secretion of STC-1 by spheroid MSCs (Sph 25k-Adh, Sph25k-Non adh, or Sph 25k DC-Adh) compared to standard monolayer culturesof MSCs (Adh High-Adh).

FIG. 28. Anti-apoptotic effects of STC-1 in cultures of RPE cells.Treatment with STC-1 (250 ng/mL) one hour following injury of ARPE-19with 450 μM H₂O₂ reduced expression of a pro-apoptotic gene (caspase3/7), cell death (Annexin V & PI staining cells) and improved cellviability (increased activity of the mitochondrial enzyme MTT).Detection of caspase activity was performed as described previously inSharma, et al., Invest. Ophthalmol. Vis. Sci., Vol. 49, No. 11, pgs,5111-5117 (2008), annexin/PI quantification as described previously inBartosh, 2010, and MTT conversion was measured as described previouslyin Mester, et al., J. Mol. Neurosci., (2010).

FIG. 29. Anti-apoptotic effect of STC-1 with intravitreal injection inRCS rats. Gene expression of BAX, a transcript that encodes apro-apoptotic protein, was reduced significantly by STC-1 as quantifiedby qRT-PCR.

FIG. 30. Immediately after chemical and mechanical injury to mousecorneas, either PBS was administered to the mice intravenously orintraperitoneally, or TSG-6 (2 mg/5 ml) was applied to the surface ofthe mouse corneas. Lateral tarsorrhaphies then were performed on theeyes of the mice. Three days later, the corneas were extracted andmyeloperoxidase (MPO) ELISA assays were performed.

FIG. 31. Early events in the cornea after injury. A. The neutrophilinfiltration occurred in the two phases: 1) a small initial phase thatbegan within about 15 min, and reached a plateau level at 4 h (Phase I)and 2) a much larger infiltration of neutrophils with a peak at 24 to 48h (Phase II). B. Based on the temporal pattern of expression inmicroarrays, the up-regulated genes in the injured cornea were dividedinto three groups. C. Real time PCR analysis of representative genes ineach group. The group A genes preceded Group B and C genes in mRNAexpression. D. Microarray heat map of genes from the corneas 4 h and 24h after injury. Gene ontology categories and the number of genesup-regulated (red) or down-regulated (blue)>2-fold are indicated. Basedon the expression pattern, genes were categorized into three groups:genes whose expression increased rapidly early after injury andthereafter decreased (Group A), genes that were expressed at steadylevels (Group B), and genes increased gradually after injury (Group C).

FIG. 32. Expression patterns of secretoneurin (SN) and HSPB4 in theinjured cornea. A, E. Western blot of SN and HSPB4 in the cornea. SN wasreleased into the cornea immediately after injury, and HSPB4 reached apeak at 4 h. B, F. G. ELISA of SN and HSPB4 in the serum and cornea. SNwas released into the cornea and the serum within 0.25 h of the injury.HSPB4 was released into the extracellular space as measured in thesupernatants of the ex vivo culture of corneas after 2 to 4 h. C, H.Immunohitochemistry of SN and HSPB4 in the cornea. D, I. Real time PCRof neuropeptides and crystallins. Among neuropeptides and crystallinsanalyzed, SN and HSPB4 showed the highest expression in the injuredcornea. J. In response to necrotic extracts, keratocytes in cultureexpressed HSPB4. K. As measured by aconitase activity, oxidative stresswas generated in the cornea by injury. L. The hydrogen peroxideincreased the expression of HSPB4 in keratocytes. M. Temporal expressionof IL-6, IL1β, CXCL1, and CCL2 in the cornea. ELISAs demonstrated thatthe expression of proteins of Group B (IL-6) and Group C (IL-1β, CXCL1,and CCL2) genes paralleled gene expression as assayed for mRNAs withmicroarrays and real time RT-PCR assays.

FIG. 33. SN reproduced the Phase I inflammatory response, and HSPB4reproduced both the Phase I and Phase II. A. The injection of therecombinant SN induced the early infiltration of neutophils of Phase I,but of Phase II. B, C. HSPB4 injection induced the Phase I and Phase IIresponses accompanied by corneal opacity and neutrophil infiltration asshown in hematoxylin-eosin staining and immunostaining for neutrophilelastase of the region of the cornea into which HSPB4 was injected. D.The topical application of the calcium channel blocker Diltiazeminhibited significantly the Phase I response in corneal injury tochemical injury. E. The subconjunctival injection of polyclonal (pAb) ormonoclonal (mAb) antibodies to HSPB4 decreased significantly theneutrophil infiltration in Phase II, compared to isotype control(IgG)-injected group. F. The amounts of SN and HSPB4 released into thecornea were dependent on the severity of injury as measured by real timePCR of SN and HSPB4 in the injured cornea and ELISA for SN and HSPB4 inthe serum or cornea. The concentrations of mRNAs and proteins werehigher in the cornea or serum by severe injury (30 sec ethanol andscraping), compared to mild injury (15 sec ethanol and scraping). G.After subconjunctival injections of clodronate-encapsulated liposome(Cl₂ MDP-LIP) on day −2 (i.e., 2 days before injury) and day 0(immediately after injury), sections of the rat cornea were stained withhematoxylin-eosin (H&E), or antibodies to CD11b and CD68 to identifymacrophages. The structure of the cornea on H&E was not affected by Cl₂MDP-LIP. CD11b- and CD68-positive cells in the cornea, however, weredecreased significantly by Cl₂MDP-LIP compared to PBS-encapsulatedliposome-injected controls (PBS-LIP).

FIG. 34. HSPB4 activated macrophages through TLR2/NK-kB signaling. A.HSPB4 did not induce the Phase II response when corneal macrophages weredepleted by subconjunctival injection of liposome-encapsulatedclondronate (Cl₂MDP-LIP). B. An intracameral injection of TSG-6, aninhibitor of TLR2/NF-kB signaling, suppressed the Phase II inflammatoryresponse of the cornea after injury. C. TSG-6 treatment also decreasedsignificantly the neutrophil infiltration in Phase II in the corneainjected with HSPB4. D. Macrophages were activated to expresspro-inflammatory cytokines when incubated with necrotic extracts of thecornea. Blocking HSPB4 with polyclonal (pAb) or monoclonal (mAb)antibodies negated significantly the effects of necrotic cornealextracts on macrophage activation. E. The addition of recombinant HSPB4activated macrophages in culture in a dose-dependent manner. F. HSPB4induced the translocation of NK-kB from the cytoplasm into nucleus inmacrophages. G. Necrotic extracts of the cornea also stimulate theTLR2/NK-kB pathway in the reporter cell expressing TLR2 (HEK-TLR2), butantibodies to HSPB4 partially inhibited the effects. H, I, J.Recombinant HSPB4 stimulated NF-kB signaling in the cell line expressingTLR2 or TLR4 (HEK-TLR4) in a dose-dependent manner, while it had noeffect in the cell without either receptor (HEK-null). K. Murinemacrophages (RAW 264.7) were incubated with Group A molecules (HSPB4,HSPB5, or β-crystallin) and evaluated for expression of pro-inflammatorycytokines by real-time RT-PCR. Neither HSPB5 nor β-crystallin activatedmacrophages, while HSPB4 induced remarkably the expression ofpro-inflammatory cytokines in macrophages. In contrast, heat-treatedHSPB4 (boiling, 20 min) did not activate macrophages in culture,indicating that HSPB4, and not contaminating pyrogens, induced themacrophage activation. Human embryonic kidney cells expressing TLR-2(HEK-TLR2) were incubated with HSPB5 or β-crystallin and evaluated foractivation of NK-kB signaling. Neither HSPB5 nor β-crystallin activatedTLR2/NK-kB signaling. L. Sterile injury was made to the rat cornea afterresident macrophages were depleted by subconjunctival injections ofclodronate-encapsulated liposome (Cl₂ MDP-LIP) on day −2 (2 days beforeinjury) and day 0 (immediately after injury). The cornea was evaluatedfor neutrophil infiltration by assays for myeloperoxidase,hematoxylin-eosin (H&E) straining, and immunostaining for neutrophilelastase to identify neutrophils. Neutrophil infiltration measured byMPO was decreased markedly in the cornea 24 hours after injury byinjection with clordronate-encapsulated liposome, compared toPBS-encapsulated liposome-injected controls (PBS-LIP). Infiltration ofinflammatory cells and neutrophils also was decreased markedly in themacrophage-depleted cornea.

FIG. 35. TSG-6 suppressed HSPB4-induced activation in macrophages in aCD44-dependant manner. A, B. TSG-6 in a dose dependent manner suppressedthe activation of macrophages by HSPB4. C, D. TSG-6 did not inhibit aHSPB4-mediated activation of NF-kB signaling in HEK-TLR2 cells which didnot express CD44. However, after the cells were transfected to expressCD44, TSG-6 dose-dependently inhibited HSPB4-mediated activation ofNF-kB signaling. E. TSG-6 inhibited significantly the inflammation inthe corneas of wild-type C57BL/6 mice, but it did not suppressinflammation in the corneas of CD44 knockout mice. F. Murine macrophages(RAW 264.7) were incubated with secretoneurin and evaluated forexpression of pro-inflammatory cytokines by real time RT-PCR.Secretoneurin did not activate macrophages. Human keratocytes werecultured with either secretoneurin or HSPB4. Neither secretoneurin norHSPB4 activated keratocytes to produce cytokines.

FIG. 36. The schematic diagram of sterile inflammation in the cornea.Immediately after injury, SN is released from nerve endings in thecornea and circulating neutrophils are recruited, thereby inducing thePhase I inflammatory response. In response to injury including oxidativestress, necrotic or injured keratocytes secrete HSPB4. The HSPB4activates resident macrophages in the cornea via the TLR2/NF-kBsignaling pathway to produce pro-inflammatory cytokines including IL-1and IL-6. These injury signals are propagated rapidly and amplified bykeratocyte activation to produce chemokines that induce neutrophilinfiltration of Phase II. TSG-6 decreased neutrophil infiltration byinhibiting the initial step of macrophage activation via TLR2/CD44/NF-kBsignaling.

DETAILED DESCRIPTION

The present invention relates to the discovery that adultstem/progenitor cells such as mesenchymal stromal cells (MSCs) possessnovel therapeutic characteristics and therefore can be useful in therapyof a desired disease such as a disease of the eye. For example, the MSCscan be used to treat diseases associated with including but not limitedto noninfectious inflammatory diseases of the cornea. This is becausethe MSCs of the invention can be manipulated to possess therapeuticcharacteristics including but not limited to expression of ananti-inflammatory protein, an anti-apoptotic protein, a protein thatregulates hematopoiesis, a protein that kills cancer cells, a proteinthat regulates immune response, a protein that regulates homing ofcells, a protein involved in cell adhesion and cell signaling, a proteinthat enhances angiogenesis, and the like.

The present invention is based on discovery of that MSCs can be used totreat inflammatory diseases of the cornea and other diseases ordisorders of the eye using intraocular injection of the recombinanttherapeutic proteins MSCs produced in response to signals from injuredtissues. This therapy is based on the discovery that after a chemicalburn to the cornea of a rat, application of MSCs or conditioned mediumfrom MSCs reduced inflammation and revascularization. In anothernon-limiting embodiment, inflammation and revascularization of thecornea can be reduced, and other diseases or disorders of the eye may betreated by application of one or more therapeutic proteins produced byactivated MSCs, such as the anti-inflammatory protein TSG-6 andbiologically active fragments or analogs thereof and/or theanti-apoptotic protein STC-1 and biologically active fragments oranalogs thereof.

Thus, in accordance with an aspect of the present invention, there isprovided a method of treating or preventing a disease or disorder of theeye in a patient. The method comprises administering to a patientmesenchymal stem cells which have been cultured under conditions toexpress an increased amount of at least one protein selected from thegroup consisting of anti-apoptotic proteins and anti-inflammatoryproteins, compared to the amount of said at least one anti-apoptoticprotein and/or anti-inflammatory protein expressed by an otherwiseidentical population of mesenchymal stem cells which have not beencultured under conditions to express an increased amount of said atleast one anti-apoptotic protein and/or anti-inflammatory protein. Themesenchymal stem cells are administered in an amount effective to treator prevent the disease or disorder of the eye in the patient.

In a non-limiting embodiment, the at least one protein is ananti-apoptotic protein. In another non-limiting embodiment, the at leastone anti-apoptotic protein is selected from the group consisting ofstanniocalcin-1 (STC-1) and stanniocalcin-2 (STC-2) and biologicallyactive fragments or analogs thereof. In yet another non-limitingembodiment, the at least one anti-apoptotic protein is STC-1 or abiologically active fragment or analogue thereof.

In another non-limiting embodiment, the at least one protein is ananti-inflammatory protein. In yet another non-limiting embodiment, theanti-inflammatory protein is TSG-6 or a biologically active fragment oranalog thereof.

In another non-limiting embodiment, the at least one anti-inflammatoryprotein may be an anti-apoptotic protein, including, but not limited to,STC-1 or a biologically active fragment or analog thereof. Although thescope of this embodiment is not to be limited to any theoreticalreasoning, unresolved inflammation may be caused in part by an increasein reactive oxygen species, or ROS. STC-1 recently has been shown toreduce reactive oxygen species by increasing expression of uncouplingprotein-2, which makes mitochondria more efficient in reducing ROS.Therefore, anti-apoptotic proteins, such as STC-1, and cells whichexpress at least one anti-apoptotic protein, including the MSCs of thepresent invention, may be useful in reducing ROS and unresolvedinflammation in the diseases and disorders of the eye mentioned herein.Thus, such anti-apoptotic proteins and the cells mentioned herein alsomay be anti-inflammatory as well as anti-apoptotic.

In another aspect of the present invention, there is provided a methodof treating or preventing a disease or disorder of the eye in a patientby administering to the patient at least one protein selected from thegroup consisting of anti-apoptotic proteins and anti-inflammatoryproteins in an amount effective to treat or prevent the disease ordisorder of the eye in the patient.

In a non-limiting embodiment, the at least one protein is ananti-apoptotic protein. In another non-limiting embodiment, theanti-apoptotic protein is STC-1 or STC-2 or biologically activefragments or analogs thereof. In yet another non-limiting embodiment,the anti-apoptotic protein is STC-1 or a biologically active fragment oranalog thereof.

In another non-limiting embodiment, the at least one protein is ananti-inflammatory protein. In another non-limiting embodiment, theanti-inflammatory protein is TSG-6 or a biologically active fragment oranalog thereof.

The present invention should not be limited only to treating disease ofthe cornea, but is applicable to any disease associated with the eye,such as diseases of the retina or the macula, including, but not limitedto, macular degeneration. That is, the invention is useful for treatingdiseases, disorders, or conditions of the eye associated withinflammation or degeneration of eye tissue.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

As used herein, the term “biocompatible lattice,” is meant to refer to asubstrate that can facilitate formation into three-dimensionalstructures conducive for tissue development. Thus, for example, cellscan be cultured or seeded onto such a biocompatible lattice, such as onethat includes extracellular matrix material, synthetic polymers,cytokines, growth factors, etc. The lattice can be molded into desiredshapes for facilitating the development of tissue types. Also, at leastat an early stage during culturing of the cells, the medium and/orsubstrate is supplemented with factors (e.g., growth factors, cytokines.extracellular matrix material, etc.) that facilitate the development ofappropriate tissue types and structures.

As used herein, the term “bone marrow stromal cells,” “stromal cells,”“mesenchymal stem cells,” “mesenchymal stromal cells” or “MSCs” are usedinterchangeably and refer to a cell derived from bone marrow (reviewedin Prockop, 1997), peripheral blood (Kuznetsov et al., 2001), adiposetissue (Guilak et al., 2004), umbilical cord blood (Rosada et al.,2003), synovial membranes (De Bari et al., 2001), and periodontalligament (Seo et al., 2005), embryonic yolk sac, placenta, umbilicalcord, skin, and blood (U.S. Pat. Nos. 5,486,359 and 7,153,500), fat, andsynovial fluid. MSCs are characterized by their ability to adhere toplastic tissue culture surfaces (Friedenstein et al.; reviewed in Owen &Friedenstein, 1988), and by being effective feeder layers forhematopoietic stem cells (Eaves et al., 2001). In addition, MSCs can bedifferentiated both in culture and in vivo into osteoblasts andchondrocytes, into adipocytes, muscle cells (Wakitani et al., 1995) andcardiomyocytes (Fukuda and Yuasa, 2006), into neural precursors(Woodbury et al, 2000; Deng et al., 2001, Kim et al., 2006; Marcsehi etal., 2006; Krampera et al, 2007). Mesenchymal stem cells (MSCs) may bepurified using methods known in the art (Wakitani et al., 1995; Fukudaand Yuasa, 2006; Woodbury et al., 2000; Deng et al., 2001; Kim et al.,2006; Mareschi et al., 2006; Krampera et al., 2007), and serve asprogenitors for mesenchymal cell lineages, including bone, cartilage,ligament, tendon, adipose, muscle, cardiac tissue, stroma, dermis, andother connective tissues. (See U.S. Pat. Nos. 6,387,369 and 7,101,704).Mesenchymal stem cells (MSCs) may be purified using methods known in theart (Wakitami, et al., 1995; Fukuda and Yuasa, 2006; Woodbury, et al.,2000; Deng, et al., 2001; Kim, et al., 2006; Mareschi, et al., 2006,Krampera, et al., 2007).

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e. increasing, decreasing, and the like.

The terms “precursor cell,” “progenitor cell,” and “stem cell” are usedinterchangeably in the art and herein and refer either to a pluripotent,or lineage-uncommitted, progenitor cell, which is potentially capable ofan unlimited number of mitotic divisions to either renew itself or toproduce progeny cells which will differentiate into the desired celltype. Unlike pluripotent stem cells, lineage-committed progenitor cellsare generally considered to be incapable of giving rise to numerous celltypes that phenotypically differ from each other. Instead, progenitorcells give rise to one or possibly two lineage-committed cell types.

“Ocular region” or “ocular site” means any area of the eyeball,including the anterior and posterior segment of the eye, and whichgenerally includes, but is not limited to, any functional (e.g., forvision) or structural tissues found in the eyeball, or tissues orcellular layers that partly or completely line the interior or exteriorof the eyeball. Specific examples of areas of the eyeball in an ocularregion include, but are not limited to, the anterior chamber, theposterior chamber, the vitreous cavity, the choroid, the suprachoroidalspace, the conjunctiva, the subconjunctival space, the episcieral space,the intracorneal space, the epicorneal space, the sclera, the parsplana, surgically-induced avascular regions, the macula, and the retina.

“Ocular condition” means a disease, ailment or condition which affectsor involves the eye or one of the parts or regions of the eye. Broadlyspeaking the eye includes the eyeball, including the cornea, and othertissues and fluids which constitute the eyeball, the periocular muscles(such as the oblique and rectus muscles) and the portion of the opticnerve which is within or adjacent to the eyeball.

“Graft” refers to a cell, tissue, organ, or otherwise any biologicalcompatible lattice for transplantation.

“Glaucoma” means primary, secondary and/or congenital glaucoma. Primaryglaucoma can include open angle and closed angle glaucoma. Secondaryglaucoma can occur as a complication of a variety of other conditions,such as injury, inflammation, vascular disease and diabetes. Theincreased pressure of glaucoma causes blindness because it damages theoptic nerve where it enters the eye. Thus, in one non-limitingembodiment, by lowering reactive oxygen species, STC-1, or MSCs whichexpress increased amounts of STC-1, may be employed in the treatment ofglaucoma and prevent or delay the onset of blindness.

“Inflammation-mediated” in relation to an ocular condition means anycondition of the eye which can benefit from treatment with ananti-inflammatory agent, and is meant to include, but is not limited to,uveitis, macular edema, acute macular degeneration, retinal detachment,ocular tumors, fungal or viral infections, multifocal choroiditis,diabetic uveitis. proliferative vitreoretinopathy (PVR), sympatheticopthalmia, Vogt Koyanagi-Harada (VKH) syndrome, histoplasmosis, anduveal diffusion.

“Injury” or “damage” are interchangeable and refer to the cellular andmorphological manifestations and symptoms resulting from aninflammatory-mediated condition, such as, for example, inflammation, aswell as tissue injuries caused by means other than inflammation, such aschemical injury, including chemical burns, as well as injuries caused byinfections, including but not limited to, bacterial, viral, or fungalinfections.

“Intraocular” means within or under an ocular tissue. An intraocularadministration of a drug delivery system includes administration of thedrug delivery system to a sub-tenon, subconjunctival, suprachoroidal,intravitreal and like location. An intraocular administration of a drugdelivery system excludes administration of the drug delivery system to atopical, systemic, intramuscular, subcutaneous, intraperitoneal, and thelike location.

“Macular degeneration related condition” refers to any of a number ofdisorders and conditions in which the macula degenerates or losesfunctional activity. The degeneration or loss of functional activity canarise as a result of, for example, cell death, decreased cellproliferation, loss of normal biological function, or a combination ofthe foregoing. Macular degeneration can lead to and/or manifest asalterations in the structural integrity of the cells and/orextracellular matrix of the macula, alteration in normal cellular and/orextracellular matrix architecture, and/or the loss of function ofmacular cells. The cells can be any cell type normally present in ornear the macula including RPE cells, photoreceptors, and capillaryendothelial cells. Age-related macular degeneration, or ARMD, is themajor macular degeneration related condition, but a number of others areknown including, but not limited to, Best macular dystrophy, Sorsbyfundus dystrophy, Mallatia Leventinese and Doyne honeycomb retinaldystrophy.

“Ocular neovascularization” (ONV) is used herein to refer to choroidalneovascularization or retinal neovascularization, or both.

“Retinal neovascularization” (RNV) refers to the abnormal development,proliferation, and/or growth of retinal blood vessels, e.g., on theretinal surface.

“Cornea” refers to the transparent structure forming the anterior partof the fibrous tunic of the eye. It consists of five layers,specifically: 1) anterior corneal epithelium, continuous with theconjunctiva; 2) anterior limiting layer (Bowman's membrane); 3)substantia propria, or stromal layer; 4) posterior limiting layer(Descemet's membrane); and 5) endothelium of the anterior chamber orkeratoderma.

“Retina” refers to the innermost of the three tunics of the eyeball,surrounding the vitreous body and continuous posteriorly with the opticnerve. It is divided into the pars optica, which rests upon thechoroids, the pars ciliaris, which rests upon the ciliary body. and thepars iridica. which rests upon the posterior surface of the iris.Grossly, the retina is composed of an outer, pigmented layer (parspigmentosa) and an inner, transparent layer (pars nervosa), whichtogether make up the pars optica. The optic part of the eye consists of9 layers named from within to outward as: 1) internal limiting membrane;2) nerve fiber layer; 3) layer of ganglion cells; 4) inner plexiformlayer; 5) inner nuclear layer; 6) outer plexiform layer; 7) outernuclear layer; 8) external limiting membrane; and 9) a layer of rods andcones.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver, Preferably, thetransplant is a human neural stem cell.

As defined herein, an “allogeneic bone marrow stromal cell (BMSC)” isobtained from a different individual of the same species as therecipient.

“Donor antigen” refers to an antigen expressed by the donor tissue to betransplanted into the recipient.

“Alloantigen” is an antigen that differs from an antigen expressed bythe recipient.

As used herein, an “effector cell” refers to a cell which mediates animmune response against an antigen. In the situation where a transplantis introduced into a recipient, the effector cells can be therecipient's own cells that elicit an immune response against an antigenpresent in the donor transplant. In another situation, the effector cellcan be part of the transplant, whereby the introduction of thetransplant into a recipient results in the effector cells present in thetransplant eliciting an immune response against the recipient of thetransplant.

As used herein, a “therapeutically effective amount” is the amount of anagent which is sufficient to provide a beneficial effect to the subjectto which the agent is administered.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced from or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnonviral compounds which facilitate transfer of nucleic acid into cells,such as, for example, polylysine compounds, liposomes, and the like.Examples of viral vectors include, but are not limited to, adenoviralvectors, adeno-associated virus vectors, retroviral vectors, and thelike.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

DESCRIPTION

The present invention relates to the discovery that MSCs express highlevels of a series of therapeutic genes such as TSG-6, for ananti-inflammatory protein and STC-1, for an anti-apoptotic protein.Accordingly, MSCs are useful as anti-inflammatory therapy in oculardiseases, including but not limited to noninfectious inflammatorydiseases of the cornea, ocular hypertension, dry eye syndrome, maculardegeneration, and retinal degeneration diseases or conditions.

The present invention encompasses methods and compositions for reducingand/or eliminating an inflammatory response in a mammal by treating themammal with an amount of MSC effective to reduce or inhibit inflammationin the eye of a mammal, and/or effect repair or healing of damagedtissue in the eye of a mammal. In one non-limiting embodiment, localapplication of MSCs, that have been preactivated in culture to expresshigh levels of therapeutic proteins to the site of injury is useful fortreating the injury. In another non-limiting embodiment, the therapeuticproteins that are secreted from the pre-activated MSCs are administeredlocally to the site of injury. In yet another non-limiting embodiment,recombinant proteins or synthetic or purified proteins, that correspondor mimic the desirable proteins secreted by the MSCs can be administeredto the site of injury.

In one non-limiting embodiment, at least one therapeutic proteinsecreted from MSCs or a recombinant, synthetic, or purified version ofthe therapeutic protein is administered to the mammal by way ofintraocular administration. For example, in a non-limiting embodiment,the MSCs or proteins may be administered intravitreally.

The present invention provides a method for treating an ocular disease,comprising injecting intraocularly into the mammal an effective amountof a therapeutic protein derived from, or expressed by MSCs or arecombinant version or a synthesized or purified version thereof, suchas TSG-6, including biologically active fragments or analogs thereof,STC-1, including biologically active fragments or analogs thereof, or acombination thereof. The invention, however, should not be limited onlyto TSG-6 and STC-1. Rather, any therapeutic protein secreted by MSCs isapplicable to the present invention of treating an ocular disease in amammal. For example, MSCs exhibit high viability and express high levelsof a series of genes for therapeutic proteins: TSG-6, for ananti-inflammatory protein; STC-1, for an anti-apoptotic protein; LIF,for a protein that regulates cell growth and development; IL-11, for aprotein that regulates hematopoiesis; TNFSF10 (also known as TRAIL), fora protein that kills some cancer cells and regulates immune response;IL-24, for a protein that kills some cancer cells; CXCR4, for a proteinthat regulates homing of cells; ITGA2 (also known as integrin α2), for aprotein involved in cell adhesion and cell signaling; and IL-8, for aprotein that enhances angiogenesis.

Ocular Injuries (A) Foreign Bodies

25% of all ocular injuries involve foreign bodies on the surface of thecornea. No scarring will occur if the injury affects only the cornealepithelium, but if it also affects the Bowman zone, scarring ispossible. After removal of the foreign body, the eye is treated with asulfonamide or antibiotic and, if there is ciliary congestion andphotophobia, or if the removal of the foreign body were difficult, it istreated with a cycloplegic such as 5% homatropine. In some instances,the therapeutic compositions of the present invention are designed toaccelerate healing of the injury caused by the foreign body and toprevent infection, and to improve the clinical outcome.

(B) Chemical Burns

Chemical burns are treated by first diluting the chemical by flushingthe eye with fluid, and then preventing infection through the use oftopical antibiotics.

Intraocular pressure may be reduced by applying timolol, epinephrine,acetazolamide. or other similar agents. If epithelialization of thecornea is incomplete after one week, there is a danger of stromalnecrosis, in addition to the risk of infection. It is therefore criticalthat the healing be accelerated to reduces these risks.

Severe scarring is another common result of chemical burns. Thetherapeutic compositions of the present invention are designed toaccelerate the healing of the corneal erosion caused by the chemicalburns, to prevent stromal necrosis and infection of the eye, and toreduce corneal scarring and thereby restore/preserve cornealtransparency.

Unexpectedly the compositions of the present invention are able toprevent or reduce scar formation while simultaneously enhancing ocularhealing, wound repair, and maintaining corneal transparency. While notwishing to be bound by any specific mechanism of action, it appears thatthese beneficial effects can be obtained due to the anti-inflammatoryactions of the compositions. In some instances, the beneficial effectscan be obtained due to the combination of anti-inflammatory andanti-apoptotic actions of the compositions of the invention.

(C) Lacerations

Lacerations of the cornea are followed by prolapse of the iris, whichcloses the injury. As in all eye injuries, there is a risk of infection.Lacerations also may extend to the sclera, which is a much more severeinjury. In such a case, surgery is required to remove prolapsed uvealtissue from the injured area, and the sclera is closed with sutures. Thetherapeutic compositions of the present invention are designed toaccelerate the healing of the laceration and to prevent infection.

(D) Traumatic, Toxic, Deficiency, and Hereditary Optic Neuropathies

Optic neuropathies affect the optic nerve, which may affect visionadversely. Traumatic optic neuropathies may be caused when the head isstruck by an object, such as a ball, or if it is pierced by an objectsuch as a bullet. Toxic optic neuropathies are caused by chemicals toxicto the optic nerve; a common example is the ingestion of methanol.Deficiency optic neuropathies can result from vitamin deficiencies suchas a B12 deficiency, and may cause lesions in the optic nerve.Hereditary optic neuropathies can be caused by mutations in the nuclearor mitochondrial genomes. The therapeutic and prophylactic compounds ofthis invention could be used to heal the optic nerve and correct vitamindeficiencies. They may also create an environment which would reduce orprevent mutations in optic cell genomes.

(E) Inflammatory Conditions

Inflammation-mediated conditions of the eye which may be treated by themethods of the invention include but is not limited to uveitis, macularedema, acute macular degeneration, retinal detachment, ocular tumors,fungal or viral infections, multifocal choroiditis, diabetic uveitis,proliferative vitreoretinopathy (PVR), sympathetic opthalmia, VogtKoyanagi-Harada (VKH) syndrome, histoplasmosis, and uveal diffusion. Ina non-limiting embodiment, the inflammation-mediated condition of theeye is uveitis. In another non-limiting embodiment, theinflammation-mediated condition of the eye is proliferativevitrioretinopathy (PVR).

Suspensions of microspheres may be used as an anti-inflammatory therapyof the eye, especially for treating inflammatory conditions of theocular adnexa, palpebral or bulbar conjunctiva, cornea and anteriorsegment of the globe. Common therapeutic applications foranti-inflammatory suspensions of microspheres include viral, allergicconjunctivitis, acne rosacea, iritis and iridocyclitis. Microspheres mayalso be used to ameliorate inflammation associated with, corneal injurydue to chemical or thermal burns, or penetration of foreign bodies. Suchconditions may result from surgery, injury, allergy or infection to theeye and can cause severe discomfort.

Notably, microspheres have considerable therapeutic advantages inreducing inflammatory responses, compared to the prevalent topicalocular use of NSAI agents and corticosteroids. Use of topical steroidsis associated with a number of complications, including posteriorsubcapsular cataract formation, elevation of intraocular pressure,secondary ocular infection, retardation of corneal wound healing,uveitis, mydriasis, transient ocular discomfort and ptosis. Numeroussystemic complications also may arise from the topical ocularapplication of corticosteroids. These complications include adrenalinsufficiency, Cushing's syndrome, peptic ulceration, osteoporosis,hypertension, muscle weakness or atrophy, inhibition of growth,diabetes, activation of infection, mood changes and delayed woundhealing.

(F) Ocular Surgical Applications

Compositions of microspheres in accordance with the present invention,may also be used to ameliorate inflammation associated with ocularsurgery, and in this context are particularly useful in a prophylacticmodality as well as in promoting healing and reducing scarring as hasbeen detailed above.

Of particular suitability is the use of the compositions of theinvention for:

post-trabeculectomy (filtering surgery); post pterygium surgery; postocular adnexa trauma and surgery; post intraocular surgery andspecifically: post vitrectomy, post retinal detachment and postretinotomylectomy.

It will be appreciated by the artisan that these are intended to serveas non-limitative examples of prevalent surgical procedures for whichthe compositions and methods of the invention are useful.

Pre-Activated

Based upon the disclosure provided herein, MSCs can be obtained from anysource. The MSCs may be autologous with respect to the recipient(obtained from the same host) or allogeneic with respect to therecipient. In addition, the MSCs may be xenogeneic to the recipient(obtained from an animal of a different species), for example rat MSCsmay be used to suppress inflammation in a human.

In a further non-limiting embodiment, MSCs used in the present inventioncan be isolated, from the bone marrow of any species of mammal,including but not limited to, human, mouse, rat, ape, gibbon, bovine. Ina non-limiting embodiment, the MSCs are isolated from a human, a mouse,or a rat. In another non-limiting embodiment, the MSCs are isolated froma human.

Based upon the present disclosure, MSCs can be isolated and expanded inculture in vitro to obtain sufficient numbers of cells for use in themethods described herein provided that the MSCs are cultured in a mannerthat promotes aggregation and formation of spheroids. For example, MSCscan be isolated from human bone marrow and cultured in complete medium(DMEM low glucose containing 4 mM L-glutamine, 10% FBS, and 1%penicillin/streptomycin) in hanging drops or on non-adherent dishes.However, the invention should in no way be construed to be limited toany one method of isolating and culturing medium. Rather, any method ofisolating and culturing medium should be construed to be included in thepresent invention provided that the MSCs are cultured in a manner thatpromotes aggregation and formation of spheroids.

Any medium capable of supporting MSCs in vitro may be used to culturethe MSCs. Media formulations that can support the growth of MSCsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimal Essential Medium (αMEM), and Roswell ParkMemorial Institute Media 1640 (RPMI Media 1640) and the like. Typically,0 to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to theabove medium in order to support the growth of MSCs. A defined medium,however, also can be used if the growth factors, cytokines, and hormonesnecessary for culturing MSCs are provided at appropriate concentrationsin the medium. Media useful in the methods of the invention may containone or more compounds of interest, including but not limited toantibiotics, mitogenic or differentiation compounds useful for theculturing of MSCs. The cells may be grown in one non-limitingembodiment, at temperatures between 27° C. to 40° C., in anothernon-limiting embodiment at 31° C. to 37° C., and in another non-limitingembodiment in a humidified incubator. The carbon dioxide content may bemaintained between 2% to 10% and the oxygen content may be maintainedbetween 1% and 22%; however, the invention should in no way be construedto be limited to any one method of isolating and culturing MSCs. Rather,any method of isolating and culturing MSCs should be construed to beincluded in the present invention.

Antibiotics which can be added into the medium include, but are notlimited to, penicillin and streptomycin. The concentration of penicillinin the culture medium is about 10 to about 200 units per ml. Theconcentration of streptomycin in the culture medium is about 10 to about200 μg/ml.

In a non-limiting embodiment, the mesenchymal stem cells are culturedunder conditions which, as noted hereinabove, provide for theaggregation of the mesenchymal stem cells into a spheroidal aggregate,and provide for optimal expression of the therapeutic protein(s).

In one non-limiting embodiment, the mesenchymal stem cells are culturedin a medium, such as complete culture medium (CCM), for example, whichincludes serum in an amount effective to upregulate one or more of thehereinabove noted therapeutic proteins. For example, the medium mayinclude fetal bovine serum in an amount of up to 20%. In a non-limitingembodiment, the fetal bovine serum is present in an amount of about 17%.The mesenchymal stem cells are cultured under conditions and for aperiod of time (for example, 7 or 8 days) sufficient to provide asufficient number of cells for further culturing. The culture medium mayinclude growth factors other than or in addition to serum to upregulateone or more of the hereinabove noted therapeutic proteins.

In one non-limiting embodiment, the spheroids can be prepared byculturing the MSCs on bacterial plates (as distinct from plates treatedfor culture of animal cells) so that the MSCs aggregate spontaneouslyinto spheroids (Bartosh, 2010).

The cells then are cultured under conditions which promote the formationof spheroidal aggregates of the cells. In one non-limiting embodiment,the cells are cultured as hanging drops. Each drop of cells containsmesenchymal stem cells in an amount which provides for optimalexpression of the at least one therapeutic protein. In a non-limitingembodiment, the hanging drops of the cells are cultured in a medium,such as complete culture medium, containing fetal bovine serum in anamount of up to 20%. In a non-limiting embodiment, the fetal bovineserum is present in an amount of about 17%.

In another non-limiting embodiment, each hanging drop of mesenchymalstem cells that is cultured contains from about 10,000 to about 500,000cells/drop. In another non-limiting embodiment, each hanging drop ofmesenchymal stem cells that is cultured contains from about 10,000 toabout 250,000 cells/drop. In a further non-limiting embodiment, eachhanging drop of cells contains from about 10,000 to about 25,000cells/drop. In yet another non-limiting embodiment, each hanging drop ofcells contains about 25,000/drop.

The hanging drops of mesenchymal stem cells are cultured for a period oftime sufficient for forming spheroidal aggregates of the mesenchymalstem cells. In general, the drops of cells are cultured for a period oftime of up to 4 days.

Once the spheroidal aggregates of the mesenchymal stem cells are formed,the mesenchymal stem cells may, if desired, be dissociated from thespheroids by incubating the spheroids in the presence of a dissociationagent, such as trypsin and/or EDTA, for example.

The invention comprises the treatment of an MSC in culture to expresstherapeutic proteins that are effective in treating a disease of theocular. For example, the MSCs can be cultured in the presence of TNF-α.In some instances, the MSCs can be pre-activated by culturing in thepresence of IFN-μγ. In other instances, the MSCs can be pre-activated byculturing in the presence of IL-1B. In some instances, MSCs can bepre-activated using any combination of TNF-α, IFN-μ, and IL-1BPre-activation of the MSCs induce the cells to secrete therapeuticproteins. Thus, the MSCs themselves, the secreted proteins, or thecombination of both provide a source of a therapeutic composition. Inone embodiment, a recombinant version of the therapeutic proteinsecreted from the pre-activated MSCs can be used as a therapeuticcomposition.

In some instances, the MSCs are contacted with an agent that inducesMSCs to secrete therapeutic proteins in a culturing medium. Theculturing medium generally comprises a base media. Non-limiting examplesof base media useful in the methods of the invention include MinimumEssential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free),F10(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with andwithout Fitton-Jackson Modification), Basal Medium Eagle (BME-with theaddition of Earle's salt base), Dulbecco's Modified Eagle Medium(DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium(GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199(M199E-with Earle's salt base), Medium M199 (M199H-with Hank's saltbase), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base),Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and MinimumEssential Medium Eagle (MEM-NAA with non essential amino acids), amongnumerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066,NCTC 135, MB 75261, MAB 8713. DM 145, Williams' G, Neuman & Tytell,Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. Apreferred medium for use in the present invention is DMEM. These andother useful media are available from GIBCO, Grand Island, N.Y., USA andBiological Industries, Bet HaEmek, Israel, among others. A number ofthese media are summarized in Methods in Enzymology, Volume LVIII, “CellCulture”, pp. 62-72, edited by William B. Jakoby and Ira H. Pastan,published by Academic Press, Inc.

Additional non-limiting examples of media useful in the methods of theinvention can contain fetal serum of bovine or other species at aconcentration of at least 1% to about 30%, preferably at least about 5%to 15%, mostly preferably about 10%.

Embryonic extract of chicken or other species can be present at aconcentration of about 1% to 30%, preferably at least about 5% to 15%,most preferably about 10%.

In a non-limiting embodiment, the MSCs are isolated from the mammal intowhich the treated MSC are to be introduced; however, the MSCs may alsobe isolated from an organism of the same or different species as themammal. The mammal may be any organism having bone tissue. In anon-limiting embodiment, the mammal is a human.

Genetic Modification

The cells of the invention may be transformed stably or transiently witha nucleic acid of interest prior to introduction into the eye of themammal. Nucleic acid sequences of interest include, but are not limitedto those encoding gene products TSG-6 and biologically active fragmentsand analogs thereof, and STC-1 and biologically active fragments andanalogs thereof. Methods of transformation of MSCs are known to thoseskilled in the art, as are methods for introducing cells into a bone atthe site of surgery or fracture.

In cases in which a gene construct is transfected into a cell, theheterologous gene is linked operably to regulatory sequences required toachieve expression of the gene in the cell. Such regulatory sequencestypically include a promoter and a polyadenylation signal.

In a non-limiting embodiment, the gene construct is provided as anexpression vector that includes the coding sequence for a heterologousprotein operably linked to essential regulatory sequences such that whenthe vector is transfected into the cell, the coding sequence will beexpressed by the cell. The coding sequence is linked operably to theregulatory elements necessary for expression of that sequence in thecells. The nucleotide sequence that encodes the protein may be cDNA,genomic DNA, synthesized DNA or a hybrid thereof, or an RNA moleculesuch as mRNA.

The gene construct includes the nucleotide sequence encoding thebeneficial protein is linked operably to the regulatory elements and mayremain present in the cell as a functioning cytoplasmic molecule, afunctioning episomal molecule, or it may integrate into the cell'schromosomal DNA. Exogenous genetic material may be introduced into cellswhere it remains as separate genetic material in the form of a plasmid.Alternatively, linear DNA which can integrate into the chromosome may beintroduced into the cell. When introducing DNA into the cell, reagentswhich promote DNA integration into chromosomes may be added. DNAsequences which are useful to promote integration may also be includedin the DNA molecule. Alternatively, RNA may be introduced into the cell.

The regulatory elements for gene expression include: a promoter, aninitiation codon, a stop codon, and a polyadenylation signal. It ispreferred that these elements be operable in the cells of the presentinvention. Moreover, it is preferred that these elements be linkedoperably to the nucleotide sequence that encodes the protein such thatthe nucleotide sequence can be expressed in the cells and thus theprotein can be produced. Initiation codons and stop codons are generallyconsidered to be part of a nucleotide sequence that encodes the protein;however, it is preferred that these elements are functional in thecells. Similarly, promoters and polyadenylation signals used must befunctional within the cells of the present invention, Examples ofpromoters useful to practice the present invention include, but are notlimited to, promoters that are active in many cells such as thecytomegalovirus promoter, SV40 promoters, and retroviral promoters.Other examples of promoters useful to practice the present inventioninclude, but are not limited to, tissue-specific promoters, i.e.promoters that function in some tissues but not in others; also,promoters of genes normally expressed in the cells with or withoutspecific or general enhancer sequences. In some non-limitingembodiments, promoters are used which express genes in the cellsconstitutively with or without enhancer sequences. Enhancer sequencesare provided in such embodiments when appropriate or desirable.

The cells of the present invention can be transfected using well knowntechniques readily available to those having ordinary skill in the art.Exogenous genes may be introduced into the cells using standard methodswhere the cell expresses the protein encoded by the gene. In someembodiments, cells are transfected by calcium phosphate precipitationtransfection, DEAE dextran transfection, electroporation,microinjection, liposome-mediated transfer, chemical-mediated transfer,ligand mediated transfer or recombinant viral vector transfer.

In some embodiments, recombinant adenovirus vectors are used tointroduce DNA with desired sequences into the cell. In some embodiments,recombinant retrovirus vectors are used to introduce DNA with desiredsequences into the cells. In other embodiments, standard CaPO₄, DEAEdextran or lipid carrier mediated transfection techniques are employedto incorporate desired DNA into dividing cells. In some embodiments, DNAis introduced directly into cells by microinjection. Similarly,well-known electroporation or particle bombardment techniques can beused to introduce foreign DNA into the cells. A second gene is usuallyco-transfected or linked to the therapeutic gene. The second gene isfrequently a selectable antibiotic-resistance gene. Standard antibioticresistance selection techniques can be used to identify and selecttransfected cells. Transfected cells are selected by growing the cellsin an antibiotic that will kill cells that do not take up the selectablegene. In most cases where the two genes co-transfected and unlinked, thecells that survive the antibiotic treatment contain and express bothgenes.

In another embodiment, the cells of the invention can be modifiedgenetically, e.g., to express exogenous genes or to repress theexpression of endogenous genes. In accordance with this embodiment, thecell is exposed to a gene transfer vector comprising a nucleic acidincluding a transgene, such that the nucleic acid is introduced into thecell under conditions appropriate for the transgene to be expressedwithin the cell. The transgene generally is contained in an expressioncassette, including a coding polynucleotide linked operably to asuitable promoter. The coding polynucleotide can encode a protein, or itcan encode biologically active RNA, such as antisense RNA or a ribozyme.Thus, the coding polynucleotide can encode a gene conferring, forexample, resistance to a toxin, a hormone (such as peptide growthhormones, hormone releasing factor, sex hormones, adrenocorticotrophichormones, cytokines such as interferons, interleukins, and lymphokines),a cell surface-bound intracellular signaling moiety such ascell-adhesion molecules and hormone receptors, and factors promoting agiven lineage of differentiation, or any other transgene with knownsequence.

The expression cassette containing the transgene should be incorporatedinto the genetic vector suitable for delivering the transgene to thecell. Depending on the desired end application, any such vector can beso employed to modify the cells genetically (e.g., plasmids, naked DNA,viruses such as adenovirus, adeno-associated virus, herpesvirus,lentivirus, papillomavirus, retroviruses, etc.). Any method ofconstructing the desired expression cassette within such vectors can beemployed, many of which are well known in the art, such as by directcloning, homologous recombination, etc. The desired vector willdetermine largely the method used to introduce the vector into thecells, which are generally known in the art. Suitable techniques includeprotoplast fusion, calcium-phosphate precipitation, gene gun,electroporation, and infection with viral vectors.

The MSCs, in a non-limiting embodiment, may have one or more genesmodified or may be treated such that the modification has the ability tocause the MSCs to self-destruct or “commit suicide” because of suchmodification, or upon presentation of a second drug (eg., a prodrug) orsignaling compound to initiate such destruction of the MSCs.

It should be understood that the methods described herein may be carriedout in a number of ways and with various modifications and permutationsthereof that are well known in the art. It should also be appreciatedthat any theories set forth as to modes of action or interactionsbetween cell types should not be construed as limiting this invention inany manner, but are presented such that the methods of the invention canbe more fully understood.

Therapy

The present invention includes a method of using MSCs as a therapy toinhibit inflammation in the context of an ocular disease. The inventionis based on the discovery that MSCs exhibit high viability and expresshigh levels of anti-inflammatory, anti-apoptotic, immune modulatory, andanti-tumorigenic molecules.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the ability of MSC to suppress inflammation andapoptosis provides a means for treating an ocular disease. In oneembodiment, the MSCs themselves can be administered to the desired siteof the eye. In another embodiment, the therapeutic proteins secretedfrom the MSCs are administered to the desired site of the eye. In yetanother embodiment, a recombinant version of the desired therapeuticprotein secreted from the MSC is administered to the site of the eye.

The invention provides a method of preventing or treating various oculardiseases or conditions, including the following: maculopathies/retinaldegeneration: macular degeneration, including age related maculardegeneration (ARMD), such as non-exudative age related maculardegeneration and exudative age related macular degeneration, choroidalneovascularization, retinopathy, including diabetic retinopathy, acuteand chronic macular neuroretinopathy, central serous chorioretinopathy,and macular edema, including cystoid macular edema, and diabetic macularedema. Uveitis/retinitis/choroiditis: acute multifocal placoid pigmentepitheliopathy, Behcet's disease, birdshot retinochoroidopathy,infectious (syphilis, Lyme Disease, tuberculosis, toxoplasmosis),uveitis, including intermediate uveitis (pars planitis) and anterioruveitis, multifocal choroiditis, multiple evanescent white dot syndrome(MEWDS), ocular sarcoidosis, posterior scleritis, serpignouschoroiditis, subretinal fibrosis, uveitis syndrome, andVogt-Koyanagi-Harada syndrome. Vascular diseases/exudative diseases:retinal arterial occlusive disease, central retinal vein occlusion,disseminated intravascular coagulopathy, branch retinal vein occlusion,hypertensive fundus changes, ocular ischemic syndrome, retinal arterialmicroaneurysms, Coat's disease, parafoveal telangiectasis, hemi-retinalvein occlusion, papillophlebitis, central retinal artery occlusion,branch retinal artery occlusion, carotid artery disease (CAD), frostedbranch angitis, sickle cell retinopathy and other hemoglobinopathies,angioid streaks, familial exudative vitreoretinopathy, Eales disease,Traumatic/surgical: sympathetic ophthalmia, uveitic retinal disease,retinal detachment, trauma, laser, PDT, photocoagulation, hypoperfusionduring surgery, radiation retinopathy, bone marrow transplantretinopathy. Proliferative disorders: proliferative vitreal retinopathyand epiretinal membranes, proliferative diabetic retinopathy. Infectiousdisorders: ocular histoplasmosis, ocular toxocariasis, presumed ocularhistoplasmosis syndrome (PONS), endophthalmitis, toxoplasmosis, retinaldiseases associated with HIV infection, choroidal disease associatedwith HIV infection, uveitic disease associated with HIV Infection, viralretinitis, acute retinal necrosis, progressive outer retinal necrosis,fungal retinal diseases, ocular syphilis, ocular tuberculosis, diffuseunilateral subacute neuroretinitis, and myiasis. Genetic disorders:retinitis pigmentosa, systemic disorders with associated retinaldystrophies, congenital stationary night blindness, cone dystrophies,Stargardt's disease and fundus flavimaculatus, Best's disease, patterndystrophy of the retinal pigmented epithelium, X-linked retinoschisis,Sorsby's fundus dystrophy, benign concentric maculopathy, Bietti'scrystalline dystrophy, pseudoxanthoma elasticum. Retinal tears/holes:retinal detachment, macular hole, giant retinal tear. Tumors: retinaldisease associated with tumors, congenital hypertrophy of the RPE,posterior uveal melanoma, choroidal hemangioma, choroidal osteoma,choroidal metastasis, combined hamartoma of the retina and retinalpigmented epithelium, retinoblastoma, vasoproliferative tumors of theocular fundus, retinal astrocytoma, intraocular lymphoid tumors.Miscellaneous: punctate inner choroidopathy, acute posterior multifocalplacoid pigment epitheliopathy, myopic retinal degeneration, acuteretinal pigment epithelitis and the like.

An anterior ocular condition is a disease, ailment or condition whichaffects or which involves an anterior (i.e. front of the eye) ocularregion or site, such as a periocular muscle, an eyelid or an eyeballtissue or fluid which is located anterior to the posterior wall of thelens capsule or ciliary muscles. Thus, an anterior ocular conditionprimarily affects or involves the conjunctiva, the cornea, the anteriorchamber, the iris, the posterior chamber (behind the retina but in frontof the posterior wall of the lens capsule), the lens or the lens capsuleand blood vessels and nerve which vascularize or innervate an anteriorocular region or site.

Thus, an anterior ocular condition can include a disease, ailment orcondition, such as for example, aphakia; pseudophakia; astigmatism;blepharospasm; cataract; conjunctival diseases; conjunctivitis,including, but not limited to, atopic keratoconjunctivitis; cornealinjuries, including, but not limited to, injury to the corneal stromalareas; corneal diseases; corneal ulcer; dry eye syndromes; eyeliddiseases; lacrimal apparatus diseases; lacrimal duct obstruction;myopia; presbyopia; pupil disorders; refractive disorders andstrabismus. Glaucoma can also be considered to be an anterior ocularcondition because a clinical goal of glaucoma treatment can be to reducea hypertension of aqueous fluid in the anterior chamber of the eye (i.e.reduce intraocular pressure).

A posterior ocular condition is a disease, ailment or condition whichprimarily affects or involves a posterior ocular region or site such aschoroid or sclera (in a position posterior to a plane through theposterior wall of the lens capsule), vitreous, vitreous chamber, retina,optic nerve (i.e. the optic disc), and blood vessels and nerves whichvascularize or innervate a posterior ocular region or site. Thus, aposterior ocular condition can include a disease, ailment or condition,such as for example, acute macular neuroretinopathy; Behcet's disease;choroidal neovascularization; diabetic uveitis; histoplasmosis;infections, such as fungal or viral-caused infections; maculardegeneration, such as acute macular degeneration, non-exudative agerelated macular degeneration and exudative age related maculardegeneration; edema, such as macular edema, cystoid macular edema anddiabetic macular edema; multifocal choroiditis; ocular trauma whichaffects a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative diabetic retinopathy), proliferativevitreoretinopathy (PVR), retinal arterial occlusive disease, retinaldetachment, uveitic retinal disease; sympathetic opthalmia; VogtKoyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocularcondition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy, photocoagulation, radiation retinopathy, epiretinal membranedisorders, branch retinal vein occlusion, anterior ischemic opticneuropathy, non-retinopathy diabetic retinal dysfunction, retinitispigmentosa, and glaucoma. Glaucoma can be considered a posterior ocularcondition because the therapeutic goal is to prevent the loss of orreduce the occurrence of loss of vision due to damage to or loss ofretinal cells or optic nerve cells (i.e. neuroprotection).

Other diseases or disorders of the eye which may be treated inaccordance with the present invention include, but are not limited to,ocular cicatricial pemphigoid (OCP), and cataracts.

In a non-limiting embodiment, when inflammation of and/or injury toand/or disease or disorder of the eye is associated with an infection,e.g., a bacterial, viral, or fungal infection, the anti-apoptotic oranti-inflammatory proteins or mesenchymal stem cells of the presentinvention may be administered in combination with at least oneanti-infective agent.

In general, at least one anti-infective agent which is administered incombination with the anti-apoptotic or anti-inflammatory proteins ormesenchymal stem cells of the present invention depends upon the type ofinfection, e.g., bacterial, viral, or fungal, to the eye, the type orspecies of bacterium, virus, or fungus associated with the infection,and the extent and severity of the infection, and the age, weight, andsex of the patient.

In a non-limiting embodiment, when the infection of the eye isassociated with one or more bacteria, at least one anti-infective agentwhich is administered in combination with the proteins or mesenchymalstem cells of the present invention is at least one anti-bacterialagent. Anti-bacterial agents which may be administered include, but arenot limited to, quinolone antibiotics, such as, for example,levofloxacin (Cravit), moxifloxacin (Vigamox), gatifloxacin (Zy-mar),cephalosporin, aminoglycoside antibiotics (e.g., gentamycin), andcombinations thereof.

In another non-limiting embodiment, when the infection of the eye isassociated with one or more viruses, the anti-infective agent which isadministered in combination with the proteins or mesenchymal stem cellsof the present invention is at least one anti-viral agent. Anti-viralagents which may be employed include those which are known to thoseskilled in the art.

In another non-limiting embodiment, when the infection of the eye isassociated with one or more fungi, the anti-infective agent which isadministered in combination with the proteins or mesenchymal stem cellsof the present invention is at least one anti-fungal agent. Anti-fungalagents which may be employed include, but are not limited to, natamycin,amphotericin B, and azoles, including fluconazole and itraconzole.

In yet another non-limiting embodiment, when the infection of the eye isassociated with more than one of bacteria, viruses, and fungi, more thanone of anti-bacterial, anti-viral, and anti-fungal agents areadministered in combination with the proteins or mesenchymal stem cellsof the present invention.

In a non-limiting embodiment, the mesenchymal stem cells or theanti-apoptotic or anti-inflammatory proteins are administered to apatient to treat or prevent macular degeneration, including the forms ofmacular degeneration hereinabove described. In another non-limitingembodiment, the mesenchymal stem cells or the anti-apoptotic oranti-inflammatory proteins may be administered to a patient incombination with other therapeutic agents employed in treating maculardegeneration. Such therapeutic agents include, but are not limited to,angiogenesis inhibitors, and anti-vascular endothelial growth factor A(VEGF-A) antibodies (eg., Avastin, Lucentis), agents or drugs which bindangiogenic agents, such as VEGF trap agents, tyrosine kinase inhibitors,which are anti-angiogenic, angiogenic protein receptor antagonists, andantibodies and antibody fragments which recognize heat shock proteins,including, but not limited to antibodies and antibody fragments whichrecognize the small heat shock protein HSPB4, HSP90, HSP70, HSP65, orHSP27, and heat shock protein antagonists, including, but not limitedto, antagonists to HSPB4, HSP90, HSP70, HSP65, and HSP27.

In accordance with another aspect of the present invention, there isprovided a method of treating or preventing a disease or disorder of theeye in a patient, comprising administering to the patient at least oneantibody or antibody fragment which recognizes a heat shock protein orat least one heat shock protein antagonist. The at least one antibody orantibody fragment or heat shock protein antagonist is administered in anamount effective to treat a disease or disorder of the eye in saidpatient.

Diseases or disorders of the eye which may be treated include, but arenot limited to, those hereinabove described.

Antibodies or antibody fragments which may be administered in accordancewith the present invention include, but are not limited to, antibodiesor antibody fragments which recognize the small heat shock proteinHSPB4, HSP90, HSP70, HSP65, or HSP27. In a non-limiting embodiment, theantibody or antibody fragment is an antibody or antibody fragment whichrecognizes the small heat shock protein HSPB4.

In another non-limiting embodiment, the mesenchymal stem cells or theanti-apoptotic or anti-inflammatory proteins of the present inventionare administered in combination with a heat shock protein antagonist. Ina non-limiting embodiment, the heat shock protein antagonist is an HSPB4antagonist.

The at least one antibody or antibody fragment may be a monoclonal orpolyclonal antibody, or a human, non-human, humanized, or chimericantibody.

The at least one antibody or antibody fragment or heat shock proteinantagonist may be administered by methods such as those hereinabovedescribed, and in conjunction with an acceptable pharmaceutical carriersuch as those hereinabove described.

Another non-limiting embodiment of the present invention encompasses theroute of administering MSCs to the recipient of the transplant. MSCs canbe administered by a route which is suitable for the placement of thetransplant, i.e. a biocompatible lattice or a donor tissue, organ orcell, to be transplanted. MSCs can be administered systemically, i.e.,parenterally, by intravenous injection or can be targeted to aparticular tissue or organ, such as bone marrow. MSCs can beadministered via a subcutaneous implantation of cells or by injection ofthe cells into connective tissue, for example, muscle. MSCs can beadministered via an intraocular implantation of cell or by injection ofcells into the desired ocular region. In a non-limiting embodiment, theMSCs are administered intracamerally, i.e., to the anterior chamber ofthe eye.

MSCs can be suspended in an appropriate diluent. Suitable excipients forinjection solutions are those that are biologically and physiologicallycompatible with the MSCs and with the recipient, such as buffered salinesolution or other suitable excipients. The composition foradministration can be formulated, produced and stored according tostandard methods complying with proper sterility and stability.

The dosage of the MSCs varies within wide limits and may be adjusted tothe individual requirements in each particular case. The number of cellsused depends on the weight and condition of the recipient, the numberand/or frequency of administrations, and other variables known to thoseof skill in the art, including, but not limited to, the age and sex ofthe patient, the disease or disorder being treated, and the extent andseverity thereof.

Administration of Therapeutic Protein

The invention provides therapeutic proteins produced by MSCs in responseto an injury signal. The therapeutic proteins can protect the cornealsurface from damage by increasing the viability and proliferation ofcorneal epithelial progenitors and by suppressing inflammation incorneal surface.

In one non-limiting embodiment, the invention provides a method fortreating or preventing a disease or disorder of the eye, such as, forexample, reducing one or more symptoms of inflammation associated withan ocular disease in a mammalian subject, comprising providing acomposition comprising purified tumor necrosis factor-alpha stimulatedgene 6 (TSG-6) protein or a biologically active fragment or analogthereof, to the mammalian subject, thereby reducing one or more symptomsof inflammation associated with an ocular disease.

In another non-limiting embodiment, the invention provides a method fortreating or preventing a disease or disorder of the eye, such as, forexample, reducing one or more symptoms of inflammation associated withan ocular disease in a mammalian subject, comprising providing acomposition comprising purified stanniocalcin-1 (STC-1) protein or abiologically active fragment or analog thereof, to the mammaliansubject, thereby reducing one or more symptoms of inflammationassociated with an ocular disease.

The therapeutic proteins of the invention can be obtained by any method.For example, in a non-limiting embodiment, the proteins can be purifiedfrom a transgenic cell that (a) comprises a heterologous nucleotidesequence encoding the protein, and (b) expresses the protein.

Any cell that may be transformed to express a heterologous nucleotidesequence may be used to express, for example, TSG-6 protein, or abiologically active fragment or analog thereof, or STC-1 or abiologically active fragment or analog thereof. Such cells include humanand non-human eukaryotic animal cells. In another embodiment, the cellis a non-human eukaryotic animal cell.

In another non-limiting embodiment, the therapeutic proteins may besynthesized by an automatic protein or peptide synthesizer by meansknown to those skilled in the art.

It will also be appreciated that fragments or variants or analogs of theabove-mentioned proteins differing in sequence from their naturallyoccurring counterparts but retaining their biological activity can alsobe used.

In certain non-limiting embodiments of the invention, a fragment orvariant or analog of a naturally occurring polypeptide is at least 70%identical, at least 80% identical, at least 90% identical, at least 95%identical, over an amino acid portion that constitutes at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90%, or 100% of the length of thenaturally occurring counterpart. For example, variant that exhibits atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, orgreater sequence identity, over the relevant portion of the sequencecould be used, wherein % identity is determined as described above. Theamino acid portion, in a non-limiting embodiment, is at least 20 aminoacids in length, and in another non-limiting embodiment, at least 50amino acids in length. Alternately, a fragment or variant can displaysignificant or, preferably, substantial homology to a naturallyoccurring counterpart. Generally a fragment or variant of a naturallyoccurring polypeptide possesses sufficient structural similarity to itsnaturally occurring counterpart that it is recognized by an antibody(e.g., a polyclonal or monoclonal antibody) that recognizes thenaturally occurring counterpart.

Administration of the compositions of this invention is typicallyparenteral, by intravenous, subcutaneous, intramuscular, orintraperitoneal injection, or by infusion or by any other acceptablesystemic method. In a non-limiting embodiment, the compositions of theinvention are provided to the mammal by intraocular administration. In anon-limiting embodiment, administration is by intravenous infusion,typically over a time course of about 1 to 5 hours. In addition, thereare a variety of oral delivery methods for the administration oftherapeutic proteins, and these can be applied to the therapeuticproteins of this invention.

Alternatively, in a non-limiting embodiment, the compositions of thepresent invention may be administered to the eye topically, such as, forexample, in the form of eye drops. In a further non-limiting embodiment,eye drops which include at least one anti-inflammatory protein, such asTSG-6 protein or an analogue or fragment thereof, are administered tothe cornea in order to treat or prevent a disease or disorder of thecornea.

In another non-limiting embodiment, the compositions of the presentinvention may be administered systemically, such as by intravenousadministration, or intraocularly, such as by intracameraladministration, i.e., to the anterior chamber of the eye.

Often, treatment dosages are titrated upward from a low level tooptimize safety and efficacy. Generally, daily dosages will fall withina range of about 0.01 to 20 mg protein per kilogram of body weight.Typically, the dosage range will be from about 0.1 to 5 mg protein perkilogram of body weight.

Various modifications or derivatives of the proteins, such as additionof polyethylene glycol chains (PEGylation), may be made to influencetheir pharmacokinetic and/or pharmacodynamic properties.

To administer the protein by other than parenteral administration, theprotein may be coated or co-administered with a material to prevent itsinactivation. For example, the protein may be administered in anincomplete adjuvant, co-administered with enzyme inhibitors oradministered in liposomes. Enzyme inhibitors include pancreatic trypsininhibitor, disopropylfluorophosphate (DEP) and trasylol. Liposomesinclude water-in-oil-in-water, CGF emulsions, as well as conventionalliposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27).

An “effective amount” of a composition of the invention is an amountthat will ameliorate one or more of the well known parameters thatcharacterize medical conditions such as inflammation associated with thecornea, as well as the other diseases and disorders of the eyehereinabove described. An effective amount, in the context ofinflammatory diseases of the cornea, as well as the other diseases ordisorders hereinabove described, is the amount of protein or cell or acombination of both that is sufficient to accomplish one or more of thefollowing: decrease the severity of symptoms; decrease the duration ofdisease exacerbations; increase the frequency and duration of diseaseremission/symptom-free periods; prevent fixed impairment and disability;and/or prevent/attenuate chronic progression of the disease.

Although the compositions of this invention can be administered insimple solution, they are more typically used in combination with othermaterials such as carriers, preferably pharmaceutical carriers. Usefulpharmaceutical carriers can be any compatible, non-toxic substancesuitable for delivering the compositions of the invention to a patient.Sterile water, alcohol, fats, waxes, and inert solids may be included ina carrier. Pharmaceutically acceptable adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. Generally, compositions useful for parenteraladministration of such drugs are well known; e.g., Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems [Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984).

Therapeutic formulations may be administered in many conventional dosageformulations. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers.

The formulations conveniently may be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. See,e.g., Gilman et al. (eds.) (1990), The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, supra, Easton, Pa.; Avis, et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, N.Y.; Lieberman et al.(eds.) (1990), Pharmaceutical Dosage Forms: Tablets, Dekker, N.Y.; andLieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: DisperseSystems, Dekker, N.Y.

In additional non-limiting embodiments, the present inventioncontemplates administration of the proteins by gene therapy methods,e.g., administration of an isolated nucleic acid encoding a protein ofinterest. The proteins of the present invention have beenwell-characterized, both as to the nucleic acid sequences encoding theproteins and the resultant amino acid sequences of the proteins.Engineering of such isolated nucleic acids by recombinant DNA methods iswell within the ability of one skilled in the art. Codon optimization,for purposes of maximizing recombinant protein yields in particular cellbackgrounds, also is well within the ability of one skilled in the art.Gene therapy methods are well known in the art. See, e.g., WO96/07321which discloses the use of gene therapy methods to generateintracellular antibodies.

There are two major approaches for introducing a nucleic acid encodingthe protein (optionally contained in a vector) into a patient's cells;in vivo and ex vivo. For in vivo delivery the nucleic acid is injecteddirectly into the patient, usually at the site where the protein isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. Commonly used vectors for ex vivodelivery of the gene are retroviral and lentiviral vectors.

Preferred in vivo nucleic acid transfer techniques include transfectionwith viral vectors such as adenovirus, Herpes Simplex I virus,adeno-associated virus, lipid-based systems (useful lipids forlipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample), naked DNA, and transposon-based expression systems. For reviewof the currently known gene marking and gene therapy protocols seeAnderson, et al., Science 256:808-8 13 (1992). See also WO 93/25673 andthe references cited therein.

Therapeutic compositions and formulations thereof of the invention canbe used, for example, for reducing inflammation due to seasonal orbacterial conjunctivitis, for reducing post-surgical pain andinflammation, to prevent or treat fungal or bacterial infections of theeye, to treat herpes ophthalmicus, to reduce intraocular pressure, or totreat endophthalmitis.

More particularly, in one non-limiting embodiment, the present inventionprovides a method for treating an ophthalmic disorder in a mammal (e.g.,including human and non-human primates), the method comprisingadministering to the eye of the mammal a therapeutically effect amountof a formulation of the present invention comprising a lipid phase, anaqueous phase and a therapeutic agent as hereinabove described, whereinthe therapeutic agent is useful for treating the ophthalmic disorder. Inone embodiment, the ophthalmic disorder is post-operative pain. Inanother embodiment, the ophthalmic disorder is ocular inflammationresulting from, e.g., iritis, conjunctivitis, seasonal allergicconjunctivitis, acute and chronic endophthalmitis, anterior uveitis,uveitis associated with systemic diseases, posterior segment uveitis,chorioretinitis, pars planitis, masquerade syndromes including ocularlymphoma, pemphigoid, scleritis, keratitis, severe ocular allergy,corneal abrasion and blood-aqueous barrier disruption. In yet anotherembodiment, the ophthalmic disorder is post-operative ocularinflammation resulting from, for example, photorefractive keratectomy,cataract removal surgery, intraocular lens implantation and radialkeratotomy.

In employing the liposome formulations of the present invention, in anon-limiting embodiment, administration is ocularly, which term is usedto mean delivery of therapeutic agents through the surface of the eye,including the sclera, the cornea, the conjunctiva and the limbus, orinto the anterior chamber of the eye. Ocular delivery can beaccomplished by numerous means, for example, by topical application of aformulation such as an eye drop, by injection, or by means of anelectrotransport drug delivery system.

In another non-limiting embodiment, the mesenchymal stem cells ortherapeutic proteins employed for treating a disease or disorder of theeye may be contained in a nanoparticle. Such nanoparticles may be formedby methods known to those skilled in the art.

Such nanoparticles may be administered ocularly, i.e., through thesurface of the eye, including the sclera, cornea, conjunctiva, and thelimbus, or into the anterior chamber of the eye. Such ocularadministration may be accomplished by any of a variety of means,including, in a non-limiting embodiment, by topical application of aformulation such as an eye drop, by injection, or by means of anelectotransport drug delivery system.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teachings providedherein.

Example 1 MSCs and MSC-Derived Factors Suppressed Inflammation andNeovascularization, and Promoted Wound Healing in Chemically-Injured RatCornea

Beneficial effects of MSCs and MSC-derived factors have been observed insuppressing corneal inflammation/neovascularization and promoting woundhealing (Oh et al., 2008). Corneal inflammation, neovascularization, anddelayed wound healing were induced in rats by applying 100% ethanol for30 sec and scraping both the epithelium in the limbus and the wholecornea. The reliability and reproducibility of this model was previouslyconfirmed and repetitively used by other researchers (Cho et al., 1998;Avila et al., 2001; Ti et al., 2002; Espana et al., 2003; Homma et al,2004; Oh et al., 2009b). Massive infiltration of inflammatory cells andgrowth of new vessels in cornea were induced in this model (FIG. 1).

Immediately after injury, rat MSCs or conditioned media (CM) derivedfrom MSC cultures were put into an applicator and allowed to remain inthe damaged cornea for two hours. A 6-mm-diameter hollow tube was usedas an applicator (FIG. 2). This method of application has previouslybeen proven effective for applying stem cells to cornea (Homma et al.,2004; Ueno et al., 2007). The following were the four groups that werestudied and subjected to different treatments: (1) 200 μL of fresh media(control group, n=10), (2) 200 μL of supernatants collected from theMSCs culture (MSC-CM I group, n=10), (3) 200 μL MSC-CM applied for twohours a day over three consecutive days (MSC-CM II group, n=10), (4) 200μL of media containing 2×10⁶MSCs (MSC group, n=10).

Effects of MSCs or MSC-CM on the cornea were determined in three ways:(I) gross examination by slit-lamp biomicroscopy based on the findingsof transparency, neovascularization (NV), and epithelial defects, (2)histological analysis for infiltration of inflammatory cells(hematoxylin-eosin staining) or CD4+ T cells (immunofluorescentstaining), and (3) ELISA and real time PCR assays for inflammation- andangiogenesis-related cytokines. As a result, it was found that cornealinflammation and NV rapidly decreased in MSC-treated corneas over timeafter injury, while they gradually increased in the vehicle-treatedcontrols (FIG. 3). The degree of corneal inflammation and NV was thelowest in the MSC group and the highest in the control group. Notably,it was observed that MSC-CM was also effective in reducing cornealinflammation and NV in proportion to the number of MSC-CM applications.More specifically, corneas treated three times with MSC-CM (MSC-CM IIgroup) were more transparent compared to both the controls and thecorneas that were treated only once with MSC-CM (MSC-CM I group).Re-epithelialization was faster in the corneas treated with MSCs andMSC-CM.

Similar to clinical findings, histological analysis (FIG. 4) revealedthat MSCs and MSC-CM II groups had fewer inflammatory cell infiltratesthan the control and MSC-CM I groups. Comparisons between MSCs andMSC-CM II groups showed that corneas treated with MSCs had significantlyfewer inflammatory cells than those treated with MSC-CM three times.

ELISA showed that production of proinflammatory cytokines IL-2 and IFN-μwas decreased in MSC- and MSC-CM-treated corneas (FIG. 5 A, B). Incontrast, large quantities of anti-inflammatory cytokines IL-10 andTGF-B were detected in MSC-treated corneas (FIGS. 5 C, D)

In order to determine the mechanisms associated with regression of newvessels by MSCs, the expression of angiogenesis-related cytokines,TSP-1, MMP-2, MMP-9, and VEGF was evaluated. Real time PCR revealed thatthe level of an anti-angiogenic factor, TSP-1, was significantlyupregulated in MSC and MSC-CM II groups (FIG. 6). The expression ofpro-angiogenic factors, MMP-2 and MMP-9, was significantly repressed inMSC-treated corneas, compared to control corneas.

Example 2 Human MSCs-Conditioned Media Rescued Human Corneal EpithelialCells from Chemically-Induced Apoptosis

Human corneal epithelial cells (hCECs) were chemically damaged byincubation in 15% ethanol for 30 seconds. Damaged hCECs were culturedwith one of the following: (1) hMSCs-conditioned media, (2) conditionedmedia from hMSCs-damaged hCECs coculture, or (3) fresh media. Then,survival of hCECs was evaluated with MTT assay. The result showed thatthe proportion of damaged hCECs was significantly decreased whencultured with hMSCs-conditioned media (FIG. 7).

Example 3 Production of MMP-9 is Significantly Suppressed inChemically-Damaged Human Corneal Epithelial Cells by hMSCs

The following experiments were performed to evaluate how MSCs affectedcorneal epithelial cells in terms of inflammatory and angiogeniccytokine secretion. The hCECs were chemically damaged, then they werecocultured with hMSCs for 24 hours, and finally the cell-freesupernatant was analyzed for cytokine concentration by ELISA. Thecoculture groups were as follows: (I) hPBMCs (human peripheral bloodmononuclear cells), (2) hCECs, (3) hPBMCs/hCECs, (4) hMSCs, (5)hMSCs/hPBMCs, (6) hMSCs/hCECs, and (7) hMSCs/hPBMCs/hCECs. As a result,it was observed that MSCs constitutively secreted VEGF, MMP-2, and TSP-1(FIG. 8). It is important to note that MMP-9, which is highly secretedby damaged hCECs, was significantly suppressed by hMSCs (FIG. 8, upperright). In fact, as a consequence of hMSC suppression the level of MMP-9was reduced from 100% to 8%. Based on these results, it was believedthat the suppression of MMP-9, a key player in corneal inflammation,angiogenesis, and wound healing would be one of the mechanismsresponsible for MSC action during corneal regeneration. MMP-9 is one ofthe pro-inflammatory proteases that has its activation significantlyinhibited by TSG-6 (Milner and Day, 2003; Milner et al., 2006).

The results presented herein demonstrate that application of either MSCsor conditioned medium from the MSCs decreased inflammation andneovascularization in a rat model for noninfectious inflammation of thecornea. Without wishing to be bound by any particular theory, thebeneficial effects of the MSCs are at least in part explained by theeffects of decreasing the levels of inflammatory cytokines andinflammation-related proteases both in the rat model of corneal damageand in cocultures with conical epithelial cells. Therefore the data areconsistent with the hypothesis that the beneficial effects of MSCs areexplained by the production by the cells of the anti-inflammatoryprotein TSG-6 and/or the anti-apoptotic protein STC-1.

Example 4 Use of Pre-Activated MSCs for the Treatment of Cornea

The following experiments were designed to test whether MSCspreactivated in culture to express therapeutic proteins would be moreeffective in reducing inflammation and neovascularization followingchemically-induced injury to the cornea than standard cultures of MSCs.The experiments were set up to compare standard preparations of MSCswith MSCs pre-activated in culture with TNFα (FIGS. 10 C and D) in therat model in terms of the minimum number of cells required to produce(a) significant improvements in neovascularization, opacity, epithelialdefects, and infiltration of inflammatory cells as in FIGS. 3 and 4; (b)significant decreases in the inflammatory cytokine IFN-μ (FIG. 5); and(c) significant decreases in the inflammation related proteases MMP-2and MMP-9 (FIG. 6). In parallel standard preparations and pre-activatedMSCs by intracameral injection (IC; into anterior chamber) in the modelare compared using the same measures of effectiveness.

As summarized in Table 1, the initial experiments are carried out withboth rat MSCs and human MSCs (hMSCs), since the hMSCs are more relevantto the potential clinical applications of the results, and human androdent MSCs have proven to be equally effective in other rodent models(Lee et al., 2009; Block et al.; 2009; Ohtaki et al., 2008; Iso et al.,2007; Ortiz et al., 2007; Lee et al., 2006; Munoz et al., 2006).

TABLE 1 MSCs Delivery Dose # Rats 1. Species hMSCs Topical 2 × 10⁴ 10 2× 10⁵ 10 2 × 10⁶ 10 rMSCs Topical 2 × 10⁴ 10 2 × 10⁵ 10 2 × 10⁶ 10 2.Route hMSCs IC 2 × 10³ 10 2 × 10⁴ 10 2 × 10⁵ 10 rMSCs IC 2 × 10³ 10 2 ×10⁴ 10 2 × 10⁵ 10 3. acMSCs Ac h/rMSCs Topical 2 × 10⁴ 10 2 × 10⁵ 10 2 ×10⁶ 10 Ac h/rMSCs IC 2 × 10³ 10 2 × 10⁴ 10 2 × 10⁵ 10 Total 180The materials and methods employed in the experiments disclosed hereinare now described.

Corneal Surface Inflammation Model

Corneal surface inflammation can be created in rats by application of100% ethanol and mechanical debridement of corneal and limbal epitheliumusing the protocol described in Oh et al. (2008). This model induces theinfiltration of neutrophils, macrophages, neovascularization, anddelayed wound healing in cornea (Figure. 1).

MSC's

Human MSCs (hMSC5) and rat MSCs (rMSCs) can be acquired fromstandardized preparations currently being distributed by our NIH/NCRRfunded center (P40 RR 17447). For hMSCs, the standardized cells can befurther screened to select preparations that are over 90% positive forPODXL. PODXL serves as a marker for MSCs that are likely to expressother epitopes (c-MET, CXCR4, and CXC3CR1) for early progenitors invivo. For rMSCs, MSCs can be isolated from the bone marrow of Lewis rats(Javazon et al., 2001).

Route of Treatment Delivery

Topical application (FIG. 2) and IC injection are compared. Thiscomparison allows for the determination of whether IC injection iseffective at lower doses than topical application. For topical delivery,a hollow tube can be applied to the cornea, the cells (200 μL) can beput into the tube, and allowed to remain in the cornea for two hours(FIG. 2). The dose of cells can be varied from 2×10⁴ to 2×10⁶, the dosepreviously found to be effective (FIGS. 3 to 6). Topical application canbe repeated on consecutive days for a total of three treatments. For ICinjection, the media (5 μL) can be injected once into the anteriorchamber of the rat eye. The dose of cells can be varied from 2×10³ to2×10⁵, i. e. to the maximal concentration that can be employed withoutaggregation of the cells (Lee et al., 2009).

Assays for Corneal Surface Regeneration

The eyes are examined with slit-lamp biomicroscopy and recorded withphotography once a week. The clinical outcome is graded by a blindedinvestigator who is an ophthalmologist under the following criteria; (1)corneal epithelial integrity, (2) transparency, and (3)neovascularization (NV). A 1% fluorescein sodium solution can be used toevaluate the degree of corneal epithelial defects. Subsequently, defectsare quantified by the ratio of epithelial defect area to total cornealarea, using an image analyzer. The corneal clarity are graded from 0 to4 using the method in Fantes et al., (1990) and Oh et al., (2008). Thecorneal NV are quantified by calculating the area of vessel growth withthe method used in D'Amato et al. (1994), Oh et al. (2008), and Oh etal. (2009b). After three weeks, corneas are excised, and examined usingappropriate assays discussed elsewhere herein.

Assays for Corneal Inflammation

Corneas are either stained with hematoxylin-eosin or subjected toimmunostaining with neutrophil- and macrophage-specific markers. Thenumbers of inflammatory cells are counted on the H&E-stained slides, Thenumbers of positively-stained cells are counted on the immunostainedslides. Also, the infiltration and accumulation of neutrophils in thecornea are quantitated by measuring myeloperoxidase (MPO) activity withthe MPO sandwich ELISA assay (Armstrong et al., 1998).

Assays for Modulation of Inflammation-, Angiogenesis-, andApoptosis-Related Molecules

The expression of proteins for inflammation-related factors (IL-1B,IL-2, IFN-μ, IL-6, IL-10, TGF-B1), angiogenesis-related factors (TSP-1,VEGF), apoptosis-related factors (Fas/Fas ligand), and gelatinases(MMP-2, MMP-9) are measured in corneas using ELISA assay.

The results of these experiments are now described. Without wishing tobe bound by any particular theory, it is believed that (1) hMSCs are aseffective in a rat model of corneal inflammation as rMSCs; (2) thepre-activated MSCs are more effective than the standard preparations ofMSCs; (3) one-time IC injection of MSCs are effective at lower dosesthan topical application.

It is possible that human proteins from hMSCs may not be as effective inthe rat model as rMSCs. However, it is believed that hMSCs are aseffective as rMSCs, because proteins are highly conserved acrossspecies, and it has been previously found that hMSCs worked in variousmurine models of inflammation (Lee et al., 2009; Block et al.; 2009;Ohtaki et al., 2008; Iso et al., 2007; Ortiz et al., 2007; Lee et al.,2006; Munoz et al., 2006). However, if it is observed that hMSCs areineffective in rats, rMSCs can be used instead of hMSCs.

MSCs activated with TNF-α may either be ineffective or may produce toxiceffects in cornea. It has been observed in preliminary experiments thatthe activation of hMSCs with TNF-α significantly upregulated somefactors to modulate inflammation and other factors to increase cellsurvival (Lee et al., 2009). However, if TNF-α activation is foundineffective, MSCs can be pre-activate with IFN-μ, IL-1B, or theircombinations as reported previously (Ren, et al., 2008). If ineffective,unmodified MSCs can be used for further experiments.

Example 5 Inflammation and Neovascularization of the Cornea can beReduced by Application of Two of the Therapeutic Proteins Produced byActivated MSCs: The Anti-Inflammatory Protein TSG-6 and theAnti-Apoptotic Protein STC-1

The following experiments were designed to assess the effectiveness ofusing TSG-6 and STC-1 for treating inflammation and neovascularizationof the cornea. Administration of recombinant proteins is an alternativeto administering MSCs to the mammal in need thereof. The experiments arecarried out as summarized in Table 2.

TABLE 2 Expt. Protein Delivery Dose # Rats 1. TSG-6 Topical  1 μg 10 10μg 10 100 μg  10 2. STC-1 Topical  1 μg 10 10 μg 10 100 μg  10 3.TSG-6 + STC-1 (1:1) Topical  1 μg 10 10 μg 10 100 μg  10 4. TSG-6 IC 0.1μg  10  1 μg 10 10 μg 10 5. STC-1 IC 0.1 μg  10  1 μg 10 6. TSG-6 +STC-1 (1:1) IC 0.1 μg  10  1 μg 10 10 μg 10 Total 180

Briefly, recombinant TSG-6 (R & D Systems, Minneapolis, Minn.),recombinant STC-1 (BioVendor Laboratory Medicine, Inc.; Czech Republic),and their combinations are applied, respectively, to rat eyes using anappropriate delivery method. If topical application is used, the dosesof TSG-6 can be varied from 1 to 100 μg (Lee et al., 2009).Alternatively, if IC injection is used, dosage from 0.1 to 10 μg can beused. The same range of doses are also tested with recombinant STC-1.The maximally effective dose of each protein are determined. Then theproteins are prepared in 1:1 mixtures and the maximally effective doseagain are determined.

Without wishing to be bound by any particular theory (I) administrationof either TSG-6 or STC-1 is effective in suppressing cornealinflammation and promoting epithelial wound healing in adosage-dependent manner; (2) alternatively, administration of 1:1mixtures may be effective at lower doses because of synergistic effectsof the proteins; (3) intracameral administration of either or bothproteins may be effective in lower doses than topical application.

Example 6 Novel Therapeutic Factors Produced by hMSCs in Response toCorneal Injury

The invention is not limited to only TSG-6 and STC-1. That is, thecurrently available data do not exclude the hypothesis that MSCs producetheir beneficial effects on tissue repair by expression of othertherapeutic genes. Therefore, experiments can be designed to test foradditional therapeutic genes expressed by MSCs in the rat model forcorneal injury. As indicated in the accompanying scheme set forth inFIG. 14, the experiments can be carried out in three complementaryphases: Phase I: use hMSCs in the rat model for corneal injury, isolatethe total RNA from the treated corneas and assay the total RNA onspecies-specific microarrays followed by filtering the data fromcross-hybridization of the probes. Phase II: repeat the in vivoexperiments using rat MSCs that express GFP (available from our NCRR/NIHcenter for distribution of MSCs), enzymatically digest the samples ofcornea, isolate the GFP-expressing rMSCs by FACS sorting, and analyzethe RNA with a rat-specific microarray. Phase III: carry out co-cultureexperiments in transwells with injured corneal epithelial cells (FIGS. 7and 8) so that the RNA from the MSCs and the target cells can beisolated separately. The microarray data from the three phases of theexperiments can be used to identify candidate therapeutic genes usingthe following criteria: (i) genes upregulated by hMSCs or rMSCs byincubation with injured cornea or corneal epithelial cells, (ii) genesfor secretory proteins, and (iii) genes whose functions suggest thatthey may have anti-inflammatory or immunosuppressive effects. Verifyingthe roles of the candidate genes can be done using real-time RT-PCR,ELISAs or Western blots, knock down of the specific genes in MSCs withsiRNAs or lentiviruses, blocking antibodies, and replacement of the MSCsby the recombinant proteins (FIGS. 10 through 13).

The materials and methods employed in the experiments disclosed hereinare now described.

Assays for hMSCs in the Rat Model for Corneal Injury

Immediately after injury, the hMSCs (2×10⁶ cells/200 μl) are placed intoan applicator and allowed to remain in the damaged cornea for two hours(FIG. 2A). Eyelids are sutured for rats not to blink in order to preventthe shedding of transplanted cells. One day, one week, two weeks, andthree weeks later, the corneas are excised, homogenized (PowerGen;Fisher Scientific), extraction of DNA (Phase Lock Gel;Eppendor/Brinkmann Instruments) and the DNA assayed for the highlyrepetitive Alu sequences, unique to the human genome, to follow the fateof hMSCs in rat eyes (FIG. 9B). The use of Alu sequences provides ahighly sensitive assay since there are about 500,000 copies per cell.However, preliminary experiments indicated that use of conventionalprocedures greatly overestimated the content of human cells. Therefore,an improved protocol based on the chemical assay for extracted DNA andstandard curves for real-time PCR of each tissue is used, The assay forAlu sequences is complemented with a less sensitive assay extracted RNA(Trizol reagent, Invitrogen) human mRNA for GAPDH in order to assay livehuman cells in the rat tissues (FIG. 9D). The results providequantitative data on the engraftment of the hMSCs.

Assays of Human and Rat mRNAs in Cornea by Microarrays

To examine the changes occurring in hMSCs applied in injured rat corneaand the changes in rat cornea caused by hMSCs, RNA will be isolated fromcornea (Trizol; Invitrogen), and assayed on both rat and humanmicroarrays (Affymetrix, Santa Clara, Calif.). Data will be filtered forcross-hybridization (Ohtaki et al., 2008; Lee et al., 2009), analyzedwith the dChip program, and normalized to a variance of 2 SD. Forcross-hybridization, we need three control groups: (i) uninjured ratcornea treated with vehicle, (ii) injured rat cornea treated withvehicle, and (iii) uninjured rat cornea treated with hMSCs. After ananalysis of human genes upregulated 2 fold or more in hMSCs, we willselect candidate genes to confirm the data by human-specific real-timeRT-PCR assays.

Real-Time PCR and ELISA Analysis

To determine whether the candidate genes arc upregulated in culturedhMSCs, real-time PCR is performed in cultured hMSCs. Double strandedcDNA is synthesized (SuperScript III; lnvitrogen) and analyzed by realtime PCR (ABI 7900, Sequence Detector; Applied Biosystems).Human-specific primers are obtained from commercial sources or designedfrom gene sequences. The data is further verified by human-specificELISA assays either from commercial sources or with kits developed fromcommercial antibodies. Alternatively, expression of the proteins areverified by Western blots where antibodies are available.

Confirmation with Recombinant Protein, siRNA, and Blocking Antibodies

Where recombinant protein is available for the candidate gene selectedabove, the protein is applied in a rat model to assess whether theprotein mimics the effects of MSCs. In addition, MSCs with a knock downof the selected gene either with an siRNA or lentivirus is tested (FIGS.11 E and F). Also, where blocking antibodies are available, they can betested in the rat model (FIG. 13).

Assays from Co-Culture in Transwells

The experiments are performed with both hMSCs and rMSCs as described inFIGS. 7 and 8. The separate cell fractions are first assayed withmicroarrays and the roles of candidate genes confirmed as above.

The results of these experiments are now described. Without wishing tobe bound by any particular theory, it is believed that: (1) one or moreof the additional cornea-protective genes can be identified from themicroarray data; (2) the protein from the candidate gene(s) can beexpressed at increased levels in the rat model and the co-cultures withinjured lens epithelium; (3) the application of the candidate protein(s)is as effective in suppressing corneal inflammation as hMSCs; (4) bothhMSCs not expressing the gene and hMSCs combined with blocking antibodyfail to produce beneficial effects on rat model.

Example 7 The Anti-Inflammatory and Anti-Apoptotic Proteins from AdultStem/Progenitor Cells (MSCs) Protect the Cornea from Injury byPreventing Apoptosis and Suppressing Inflammation

The following experiments were designed to determine whetherinflammation of the cornea can be reduced by application of two of thetherapeutic proteins produced by activated MSCs: the anti-inflammatoryprotein TSG-6 and/or the anti-apoptotic protein STC-1.

To test the anti-apoptotic effect of STC-1 in vitro, human cornealepithelial progenitor cells (hCEPs) after exposure to ethanol werecultured for 24 hours with one of the following: (a) conditioned mediumfrom standard cultures of human MSCs (hMSCs), (b) conditioned mediumfrom hMSCs pre-activated to express therapeutic factors by incubationwith TNF-α for 24 hr, (c) rhSTC-1, (d) conditioned medium and blockingAb against rhSTC-1, or (e) IgG. Both fresh medium and the mediumconditioned from cultures of human dermal fibroblasts were used ascontrols. After incubation, the cell viability, proliferation andapoptosis were evaluated using MTT assay, BrdU uptake, and PI/annexinflow cytometry. It was observed that conditioned medium frompre-activated MSCs and rhSTC-1 had the greatest effects in hMSCsimproving the viability, increasing the proliferation, and inhibitingthe apoptosis of damaged hCEPs (FIG. 15).

To test the anti-inflammatory effect of TSG-6 in vivo, corneal surfaceinflammation was created in Lewis rats by ethanol application andmechanical debridement of corneal and limbal epithelium. Immediatelyafter injury, rhTSG-6 (2 ng-2 ug) or the same volume of PBS was injectedinto the anterior chamber. Effects on the cornea were determined inthree ways: (1) gross examination by slit-lamp biomicroscopy based onthe findings of transparency and neovascularization (NV), (2)histological analysis for infiltration of inflammatory cell(hematoxylin-eosin staining), (3) myeloperoxidase (MPO) assay forinfiltration of neutrophils, (4) ELISA and real time PCR forinflammation-related chemokines and cytokines, (5) Gel zymography andELISA for total and active MMP-9. Changes in inflammatory markers insystemic circulation were also determined by WBC counting and serum MPGevaluation. It was observed that corneal opacity and neovascularizationwere significantly decreased in TSG-6-treated corneas compared toPBS-treated controls. (FIG. 16). The infiltration of inflammatory cellsand expression of MMP-9 were significantly decreased in TSG-6-treatedcorneas compared to PBS-treated controls. (FIG. 17). It was alsoobserved that TSG-6 up to the concentration of 0.002 ug/ml was effectivein reducing corneal opacity, inflammation, and MMP-9 production (FIG.19). Furthermore, it was observed that administration of TSG-6 decreasedthe expression of inflammatory cytokines and chemokines (FIGS. 20, 21,22).

The results presented herein demonstrate that therapeutic proteinsproduced by MSCs in response to an injury signal can protect the cornealsurface from damage by increasing the viability and proliferation ofcorneal epithelial progenitors and by suppressing inflammation incorneal surface.

Example 8 1. Potential of Adult Stem/Progenitor Cells (MSCs) and STC-1as Anti-Apoptotic Therapies in Degenerative Diseases of the Retina

1.1. MSCs Rescue Degenerative Diseases of the Retina.

Bone marrow-derived rodent MSCs have been transplanted via subretinalinjection in mouse and rat models of inherited retinal degenerations(Pan, et al., Acta Biochem. Biophys. Sin (Shanghai), Vol. 40, No. 3,pgs. 202-208 (2008); Inoue, et al, Exp. Eye Res., Vol. 85, No. 2, pgs.234-241 (2007); Arnhold, et al., Graefe's Arch. Clin. Exp. Ophthalmol.,Vol. 245, No. 3, pgs. 414-422 (2007); Kicic, et al., J. Neurosci, Vol.23, No. 21, pgs. 7742-7749 (2003)). Anatomic preservation of the ONL wasdemonstrated by histologic analysis (Pan, 2008; Inoue, 2007; Arnhold,2007). In addition, electroretinography (Inoue, 2007) demonstratedpreservation of retinal function with MSC therapy. These publishedresults using subretinally delivered rodent MSCs suggest a positivetherapeutic effect for MSCs in the rescue of retinal degeneration.Although some studies emphasized the production of secreted factors(Inoue, 2007), the mechanism of action or secreted factors were notdefined. Additionally, subretinal delivery of cell therapy has importantdrawbacks: retinal detachment following subretinal cell therapy (Pan,2008) and the potential for only local rescue of photoreceptors at theinjection site (Arnhold, 2007).

1.2. MSCs can Rescue Tissues by Secretion of the Anti-Apoptotic ProteinSTC-1.

The therapeutic benefit of adult stem/progenitors from bone marrow(mesenchymal stem cells, marrow stromal cells, MSCs) in various diseasemodels has been known for some time (Prockop, Science, Vol. 276, pgs.71-74 (1997); Prockop, et al; Proc. Nat. Acad Sci., Vol. 100, Supp. 1,pgs. 11917-11923 (2003)). The cells can engraft and differentiate toreplace injured cells but more frequently they produce functionalimprovements by being activated by signals from injured tissues toexpress therapeutic factors (Lee, Cell Stem Cell (2009)). Among thetherapeutic factors produced by MSCs is the anti-apoptotic proteinstanniocalcin-1 (STC-1). STC-1 was identified by our lab in experimentsthat tested the hypothesis that soluble factors secreted by MSCs couldreduce apoptosis in an in vitro model of UV-irradiated skin fibroblasts.It was found that fibroblast apoptosis was reduced by transwellco-culture of MSCs. Microarray analysis detected increased expression ofthe anti-apoptotic protein Stanniocalcin-1 (STC-1) by MSCs, a findingthat was later confirmed by Western Blot analysis. The resultsadditionally were demonstrated when addition of an antibody againstSTC-1 inhibited the ability of MSCs to reduce apoptosis of UV-irradiatedfibroblasts (Block, et al., Stem Cells, Vol. 27, No. 3, pgs. 670-681(2009)). Additionally, recombinant human STC-1 was able to reducehypoxia induced apoptosis in A549 cells (Block, 2009). One reportedaction of STC-1 that explains the anti-apoptotic and anti-necroticeffects of STC-1 is the uncoupling of oxidative phosphorylation thatleads to a decrease in ROS production via induction of mitochondrialuncoupling protein 2 (UCP2) (Wang, et al., J. Leukoc. Biol., Vol. 86,No. 4, pgs. 981-988 (2009)). Our collaborators also have confirmedrecently that the anti-apoptotic effects of STC-1 seen in a lung cancercell line are due to a UCP2 dependent ROS reduction (Ohkouchi et al,Proc. Nat. Acad. Sci., in press). STC-1 also has been shown to beupregulated in neurons in response to cerebral ischemia in both rats andhumans (Zhang, et al., Proc. Nat. Acad. Sci., Vol. 97, No. 7, pgs.3637-3642 (2000)) and to protect neuronal cells from hypoxia in vitro(Zhang, 2000). Other actions of STC-1 have been described, which includeits ability to inhibit inflammation (Sheik-Hamad, et al., Am. J.Physiol. Renal. Physiol., Vol. 298, No. 2, pgs. 248-254 (2010)) byinhibiting macrophage chemotaxis (Kanallis, et al., Am. J. Physiol.Renal Physiol. Vol. 286, No. 2, pgs. 356-362 (2004)), modulatingtransendothelial migration of leukocytes (Chakraborty, et al., Am. J.Physiol. Renal Physiol., Vol. 292, No. 2, pgs. 895-904 (2007)), andreducing T-cell infiltration (Huang, et al., Am. J. Pathol., Vol. 174,No. 4, pgs. 1368-1378 (2009)). These observations suggested that thetherapeutic benefits of MSCs observed previously in models of retinaldegeneration (Pan, 2008; Inoue, 2007; Arnhold, 2007) were explained bythe cells' secreting STC-1 to decrease the apoptosis that is a prominentfeature of the pathology in both animal models and patients (Dunaief,2002; Xu, 1996; Cottet, 2009; Doonan, 2004, Yu, 2004; Katai, 1999;Katai, 2006).

This experiment employs (a) intravitreal administration of theanti-apoptotic protein STC-1 and human adult stem/progenitor cells thatcan be obtained readily from bone marrow to reduce photoreceptor deathin the degenerating retina; and (b) a battery of highly sensitive assaysand experimental protocols that have been used previously in order toelucidate the molecular and cellular mechanisms whereby MSCs producetheir therapeutic benefits in other animal models of human diseases.

Approach

Aim 1. Test the Hypothesis that we can Improve Our Preliminary ResultsUsing Intravitreal Administration of STC-1 in the Royal College ofSurgeons (RCS) Rat Model of Retinal Degeneration by Optimizing the Dose,Time, and Frequency of Administration of STC-1.

1.1. Rationale.

To determine whether intravitreal administration of STC-1 delaysphotoreceptor degeneration in vivo, two rodent models of retinaldegeneration were used. S334ter-3 rhodopsin transgenic rats were treatedat post natal day 9 (P9) and again at P12 with intravitreal injectionsof 1 μg STC-1. The rats were sacrificed at P19. Histologic analysisrevealed increased outer nuclear (ONL) thickness compared to uninjected(UI) controls. Mean ONL thicknesses of inferior (inf), superior (sup),and total retina were quantified as described previously in Lewin, etal., Nat. Med. Vol. 4, No. 8, pgs. 967-971 (1998). Total retina ONLthickness was increased significantly in STC-1 treated rats (n=4,p=0.018) (FIG. 23).

Additionally, the Royal College of Surgeons (RCS) rat model of retinaldegeneration was tested. As part of these studies, a gene expressionassay was developed that could be used in conjunction with histologicand functional analysis to detect photoreceptor rescue. Expression ofphotoreceptor specific genes was tested by quantitative real-time PCR(qRT-PCR) in retinas isolated from RCS rats at varying timepoints.Photoreceptor gene expression was shown to decrease overtime in the RCSrat in rates comparable with the previously described decline of the ONLin LaVail, et al., Exp. Eye Res., Vol. 21, No. 2, pgs. 167-192 (1975)(FIG. 24).

Utilizing this assay as a basis for quantification of photoreceptorviability, the hypothesis that intravitreal administration of STC-1would improve photoreceptor viability was tested. Rats received anintravitreal administration of 2.5 μg STC-1. Injections occurred at P21,the approximate time of initiation of ONL decline (LaVail, 1975). Therats were sacrificed, and tissue was collected at P40. Total RNA fromthe retinas of the rats was extracted using the RNeasy mini kit.(Qiagen). cDNA was generated by reverse transcription (Super Script III;Invitrogen) using 1 μg total RNA. Real time amplification was performedusing the Taq Man Universal PCR Master Mix (Applied Biosystems). An 18SrRNA probe (Taq Man) was used for normalization of gene expression. Geneexpression analysis showed significant increases in photoreceptor geneexpression in STC-1 treated animals compared to PBS injected controls.(FIG. 25).

1.2. Design.

Intravitreal administration of STC-1 reduced photoreceptor degenerationin two rodent models of retinal degeneration. As described hereinbelow,the next set of experiments are directed to optimizing the dose, time ofadministration, and frequency of administration of STC-1 in the RCS rat.

1.2.1. Optimization of Dose of STC-1.

First, the dose of STC-1 which preserves photoreceptor viability mosteffectively (STC-1^(PotD)) is determined. The experiments are carriedout as summarized in Table 3. For this experiment, STC-1 is injected atP21 and tissues are harvested at P40. The results are assessed on thebasis of photoreceptor gene expression (see FIG. 24).

TABLE 3 Dose of STC-1. P21, postnatal day 21; P40, post-natal day 40.qRT-PCR, for photoreceptor genes as described in FIG. 24.; fellow eye,indicates the fellow (contralateral) eye is injected as an internalcontrol. No. Therapy Dose (μg) Injection Termination Evaluations (RCSRat) 2.5 P21 P40 qRT-PCR 6 1.0 P21 P40 qRT-PCR 6 STC-1 0.5 P21 P40qRT-PCR 6 0.1 P21 P40 qRT-PCR 6 0.05 P21 P40 qRT-PCR 6 PBS 0 P21 P40qRT-PCR fellow eye Milestone: Optimal dose of STC-1 = STC-1^(OptD).

In parallel Experiments, as outlined in Table 4 below, the optimal timeof administration will be defined and whether two injections are moreeffective than one will be determined.

TABLE 4 Day of injection and number of treatments. Injection TherapyDose 1^(st) inj. 2^(nd) inj. Termination Evaluations No. (RCS Rat) STC-1STC-1^(OptD) P14 none P40 qRT-PCR 6 STC-1^(OptD) P14 P21 P40 qRT-PCR 6STC-1^(OptD) P21 P28 P40 qRT-PCR 6 STC-1^(OptD) P28 none P40 qRT-PCR 6STC-1^(OptD) P28 P35 P40 qRT-PCR 6 PBS 0 fellow eye P40 qRT-PCR felloweye Milestone: Optimal time of administration of STC-1 = STC-1^(OptT).

1.2.2. Histological and Functional Therapeutic Rescue by STC-1^(OptD)and STC-1-^(OptT).

Histologic and functional tests are used to test the ability ofSTC-1^(OptD) and STC-1^(OptT) to rescue retinal degeneration compared toPBS injected controls. Fixed eyes are sent for quantification of ONLthickness as described in Lewin, et al., 1998. Electroretinogram (ERG)analysis is performed as described previously in Ren, et al., Exp. EyeRes.; Vol. 70, No. 4, pgs. 467-473 (2000).

TABLE 5 Histologic and functional tests of photoreceptor rescue. No.(RCS Therapy Dose Injection(s) Termination Evaluations Rat) STC-1STC-1^(OptD) STC-1^(OptT) P40 Histology 6 STC-1 STC-1^(OptD)STC-1^(OptT) P40 ERG 6 PBS 0 P40 fellow eye Milestone: Anatomic andfunctional measurements of the ability of STC-1 to rescue retinaldegeneration.

Aim 2. Test the Hypothesis that Intravitreal Administration of MSCsPre-Activated in Culture to Secrete Large Amounts STC-1 Will ProvideImprovement of Photoreceptor Viability on the Basis that SustainedSecretion of STC-1 Will be More Effective than Protein AdministrationAlone.

2.1. Rationale.

In order to determine whether cell therapy may provide greater rescue ofphotoreceptor viability than protein therapy alone, the hypothesis thatintravitreally administered human MSCs can survive in the rat vitreouscavity was tested. Vials of frozen passage one human mesenchymal stemcells (hMSCs) were obtained from the Center for the Preparation andDistribution of Adult Stem Cells(http://medicine.tamhsc.edu/irm/msc-distribution.html). Following 24hour recovery, the hMSCs were plated at a density of 100 cells/cm² andincubated at 37° C. in complete culture medium (CCM) with 16% fetalbovine serum (FBS) for 8 days until approximately 70% confluence wasreached. Passage three cells were used for all experiments.

An intravitreal injection of 1×10⁵ human MSCs was performed atpost-natal day 21. Four days following injection the eye was enucleatedand RNA was extracted from the whole globe to evaluate how many humancells remained in the eye cavity. Expression of human GAPDH (hGAPDH) wasmeasured by qRT-PCR as hereinabove described, thereby providing anindication of any surviving human cells in the rat eye. Based on thestandard curve generated (FIG. 26, Left Panel), an estimated 10-20% ofinjected cells remained viable for four days following injection (FIG.26, Right Panel). This data provides the basis to test the hypothesisthat sustained secretion of STC-1 by MSCs pre-activated in culture tosecrete large amounts of STC-1 would have a greater therapeutic benefitthan administration of protein alone. Additionally, other secretedfactors from MSCs including the anti-inflammatory protein TSG-6 (Lee,2009), neurotrophic factors (Li, et al., Graefe's Arch. Clin. Exp.Ophthalmal., Vol. 247, No. 4, pgs. 503-514 (2009)) such as CNTF, bDNF,or bFGF, or the retinal-protective protein LIF (Bartosh, et al., Proc.Nat. Acad. Sci., Vol. 107, No. 31, pgs. 13724-13729 (2010); Joly, et al;J. Neurosci., Vol. 28, No. 51, pgs. 13765-13774 (2008)) may act inconjunction with STC-1 to preserve photoreceptor viability.

MSCs in standard culture conditions express relatively low levels oftherapeutic proteins unless stimulated in culture or activated in vivoby injury signals from the host (Lee, 2009; Bartosh, 2010). A recentreport from our laboratory demonstrated that culturing MSCs as 3Dspheroids activates the cells to produce large amounts of therapeuticmolecules including STC-1 (Bartosh, 2010). Compared to standard culturepreparations of MSCs, culture of the cells as 3D spheroids of 25,000cells (Sph 25K) enhanced secretion of STC-1 about 20-fold. Spheroids canbe dissociated into MSC spheroid dissociated cells (Sph 25k DC) whichretain the ability to secrete high levels of STC-1 compared to monolayerMSCs (Adh High). (FIG. 27).

2.1.1. Optimization of the Dose of Sph 25k DCs.

First, the dose of Sph 25k DCs which preserves photoreceptor viability(Sph 25k DC^(OptD)) most effectively is determined. The experiments arecarried out as summarized in Table 6. For this experiment, Sph 25k basedon photoreceptor gene expression (see FIG. 24) are injected.

TABLE 6 Dose of Sph 25k DCs. qRT-PCR analysis for. photoreceptor genesas described in FIG. 24. No. Dose (RCS Therapy (×10³ cells) InjectionTermination Evaluations Rat) Sph 25k 100 P21 P40 qRT-PCR 6 DCs 50 P21P40 qRT-PCR 6 10 P21 P40 qRT-PCR 6 MSCs 100 P21 P40 qRT-PCR 6 50 P21 P40qRT-PCR 6 10 P21 P40 qRT-PCR 6 Fbs 100 P21 P40 qRT-PCR 6 50 P21 P40qRT-PCR 6 10 P21 P40 qRT-PCR 6 PBS 0 P21 P40 qRT-PCR fellow eyeMilestone: Optimal dose of Sph 25k DCs = Sph 25k DC^(OptD).

Potential Pitfalls and Alternative Strategies.

If MSCs provide greater rescue than Sph 25k DCs, then MSCs are used insubsequent experiments.

2.1.2. Optimization of the Administration Time and Frequency of Sph 25kDCs.

In parallel experiments, carried out as summarized in Table 7 below, theoptimal time of administration is defined and whether two injections aremore effective than one is determined.

TABLE 7 Time and frequency of administration of Sph 25k DCs. InjectionTherapy Dose 1^(st) inj. 2^(nd) inj. Termination Evaluations No. (RCSRat) Sph 25k SPH 25k DC^(OptD) P14 none P40 qRT-PCR 6 DCs SPH 25kDC^(OptD) P14 P21 P40 qRT-PCR 6 SPH 25k DC^(OptD) P21 P28 P40 qRT-PCR 6SPH 25k DC^(OptD) P28 none P40 qRT-PCR 6 SPH 25k DC^(OptD) P28 P35 P40qRT-PCR 6 PBS 0 fellow eye P40 qRT-PCR fellow eye Milestone: Optimaltime of administration of Sph 25k DCs = Sph 25k DC^(OptT).

2.1.3. Histological and Functional Therapeutic Effects of Sph 25kDC^(OptD) and Sph 25k DC^(OptT).

Histologic and functional tests carried out as described in Table 8below, are used to test the ability of Sph 25k DC^(OptD) and Sph 25kDC^(OptT) to rescue retinal degeneration compared to PBS control.

TABLE 8 Histologic and functional tests of photoreceptor rescue. TherapyDose Injection Termination Evaluations No. (RCS Rat) Sph 25k DCs Sph 25kDC^(OptD) Sph 25k DC^(OptT) P40 Histology 6 Sph 25k DC^(OptD) Sph 25kDC^(OptT) P40 ERG 6 PBS 0 P40 Histo/ERG fellow eye Milestone: Anatomicand functional evidence of the rescue effects of Sph 25k DCs.

Aim 3. Test the Hypothesis that Our Preliminary Results DemonstratingRescue of Photoreceptors in the RCS Rat are Explained by PreviousObservations that STC-1 Inhibits Apoptosis and Reduces Reactive OxygenSpecies by Uncoupling Oxidative Phosphorylation.

3.1 Rationale.

It has been proposed that patients with RP undergo cone photoreceptordeath due to oxidative damage following rod photoreceptor death (Shen,2005; Usui, Mol. Ther., Vol. 17, No. 5, pgs. 778-786 (2009)). Increasedlevels of oxygen have been observed in rodent models of RP including theRCS rat as photoreceptor degeneration occurs (Yu, 2004; Yu, et al.,Invest. Ophthalmol. Vis. Sci., Vol. 41, No. 12 pgs. 3999-4006 (2000)).As a result of increased levels of oxygen, oxidative damage tophotoreceptors has been observed both in small (Komeima, 2006) and large(Shen, 2005) animal models of RP. Further evidence includes studies thatdemonstrate antioxidant therapy slows photoreceptor death in animalmodels of RP (Komeima, 2007). The hypothesis was tested initially in anin vitro model of oxidative RPE injury. Human RPE cells from cell lineARPE-19 (ATCC Catalog No. CRL-2302), were damaged with 450 μM hydrogenperoxide as described previously in Kim, et al., Korean J. Ophthalmol.,Vol. 17, No. 1, pgs. 19-28 (2003). As summarized in Table 9 below, onehour after injury, the cells were treated with 250 ng/ml STC-1, or witha vehicle (control).

TABLE 9 In vitro study using STC-1 treatment of hydrogenperoxide-damaged ARPE-19. Therapy Dose Assays STC-1 250 ng/ml TUNEL,mito. potential, lactate production, qRT-PCR Vehicle TUNEL, mito.potential, lactate production, qRT-PCR

The cells, following treatment, were evaluated for expression of apro-apoptotic gene (caspase 3/7), cell death (Annexin V & PI staining ofcells) and improved cell viability (increased activity of themitochondrial enzyme MTT). Detection of caspase activity was performedas described in Sharma, et al., Invest. Ophthalmol. Vis. Sci., Vol. 49,No. 11, pgs. 5111-5117 (2008), annexin/PI quantification as described inBartosh, et al., Proc. Nat. Acad. Sci., Vol. 107, No. 31, pgs.13724-13729 (2010), and MTT conversion was measured as describedpreviously in Mester, et al., J. Mol. Neurosci. (2010).

Following injury, it was observed that treatment with STC-1 reducedapoptosis and improved cell viability compared to vehicle controls.(FIG. 28).

Additionally, the hypothesis was tested in the RCS rat. Following anintravitreal injection of 2.5 μg STC-1 in the RCS rat at P21, geneexpression of BAX, a transcript that encodes a pro-apoptotic protein, isreduced significantly in the RCS retina at P40 (FIG. 29) as assessed byqRT-PCR. Therefore, these results are consistent with the idea thatSTC-1 inhibits the RCS Retina apoptosis. The hypothesis that the resultsare because of the ability of STC-1 to reduce reactive oxygen species byuncoupling oxidative phosphorylation then is tested.

3.2. Design.

In vitro experiments are carried out as summarized in Table 9. In vivoexperiments are carried out as summarized in Table 10. First, the invitro model of hydrogen peroxide induced ARPE-19 injury (Kim, 2003) isused. Apoptosis in vitro is evaluated using TUNEL stain, lactateproduction (Tanito, Invest. Ophthalmol. Vis. Sci., Vol. 46, No. 3, pgs.979-987 (2005)), and qRT-PCR. The level of reactive oxygen species, orROS, is evaluated using measurements of mitochondrial potential. Changesin UCP2 with qRT-PCR also is evaluated.

For in vivo studies, apoptosis in the retina of RCS rats is evaluatedusing TUNEL stain (Mizukoshi, Exp. Eye Res., Vol. 91, No. 3, pgs.353-361 (2010)) and qRT-PCR. In addition, levels of ROS are evaluated bymeasuring tissue aconitase activity as described previously in Tarpey,et al., Am. J. Physiol. Regul. Integr. Comp. Physiol., Vol. 286, No. 3,pgs. 431-444 (2004).

TABLE 10 In vivo study using intravitreal administration of STC-1 in theRCS rat. No. (RCS Therapy Dose Injection(s) Termination Assays Rats)STC-1 STC-1^(OptD) STC-1^(Optl) P40 TUNEL, 6 aconitase, qRT-PCR VehicleP40 TUNEL, 6 aconitase, qRT-PCR

Example 9

Fifteen mice were anesthetized by isoflurane inhalation. In order tocreate a chemical burn to the cornea, 100% ethanol was applied to thewhole cornea including the limbus for 30 seconds, followed by rinsingwith 10 ml of balanced salt solution. The whole cornea and limbalepithelium then were scraped mechanically using a surgical blade. (SeeOh, et al., Proc. Nat. Acad. of Sci., Vol. 107, No. 39, pgs. 16875-16880(Sep. 28, 2010)). Two mice served as controls. Immediately thereafter, 5μl of PBS were administered intravenously or intraperitoneally to 10mice, and 2 μg/5 μl of TSG-6 were applied topically to the cornealsurfaces of 5 mice. The eyelids of the mice then were closed with one8-0 silk suture at the lateral third of the lid margin.

Three days later, the mice were killed and the corneas were excised. Thecorneas then were sectioned into small pieces and lysed in 150 μl oftissue extraction reagent containing protease inhibitors (Invitrogen).The samples then were sonicated on ice and centrifuged twice (15,000×gat 4° C. for 20 minutes). The supernatants were assayed with commercialELISA kits for myeloperoxidase (MPO) (MPO ELISA kit, Hy Cult Biotech).

As shown in FIG. 30, topical administration of TSG-6 suppressed cornealinflammation more effectively than the controls.

Example 10

Sterile inflammation now is recognized to play a key role in manydiseases that include myocardial infarction, stroke, Alzheimer'sdisease, and atherosclerosis (Chen, et al., Nat. Rev. Immunol., Vol. 10,No. 12, pgs. 826-837 (2010); Rock, et al., Ann. Rev. Immunol., Vol. 28,pgs. 321-342 (2010); Spite, et al., Circ. Res., Vol. 107, No. 10, pgs.1170-1184 (2010)). The molecular and cellular responses of sterileinflammation include over 20 nonmicrobial endogenous stimuli referred toas damage-associated molecular patterns (DAMPs) which signal throughpattern recognition receptors (PRRs) on resident macrophages thatactivate at least three intracellular pathways to upregulate theexpression of pro-inflammatory cytokines. In spite of the intenseinterest in the field, a series of important questions remainunanswered, including whether some DAMPs identified from roles in vitroplay important roles in vivo, whether some DAMPS play redundant roles,and whether different tissues used different DAMPs (Matzinger, Nat.Immunol., Vol. 8, No. 1, pgs. 11-13 (2007)).

The cornea is an attractive model system to investigate sterileinflammation because it is accessible readily to experimentalmanipulations in vivo and in vitro. Moreover, sterile inflammationoccurs in diseases of the cornea that include limbal stem celldeficiency, chemical burns, and allergic or autoimmune keratitis(Wagner, Surv. Ophthalmol., Vol. 41, No. 4, pgs. 275-313 (1997);Krachmer, Cornea, 2^(nd). Ed., Vol. 1, pgs 1179-1308). To examine thetemporal sequence and stimuli for sterile inflammation, a model was usedin which the cornea was injured by exposure to alcohol followed byscraping to remove the epithelium of the cornea and limbus that containsstem cells. It was observed that the injury provoked two distinct phasesof neutrophil infiltration: A small initial Phase I that began in 15 mmand reached a plateau between 4 to 8 hours and a much larger secondPhase II that peaked at 24 hours to 48 hours. Analysis of the two phasesdemonstrated that Phase I was stimulated by the neuropeptidesecretoneurin and perhaps other signals. The second, more massive PhaseII of neutrophil infiltration was simulated by a small heat shockprotein, HSPB4, that was synthesized and released in injured keratocytesof the corneal stroma and that acted as a DAMP to activate residentmacrophages.

Methods Animals

Lewis rats (LEW/Crl) were purchased from Charles River Laboratory(Wilmington, Mass.). HSPB4 knockout mice (Cryaa⁻¹⁻) were generatedoriginally at the National Eye Institute by targeted gene disruption andwere maintained in the 129 S6/SvEvTac background (Brady, et al. Proc.Nat. Acad. Sci., Vol. 94, No. 3, pgs. 884-889 (1997). 129S6/SvEvTac,C57BL/6 (C57BL/6J) and CD44 knockout mice (CD44h⁻¹⁻; B6.Cg-Cd44tm1Hbg/J)were purchased from the Jackson Laboratory (Bar Harbor, Me.). Allanimals were used under a protocol approved by the Institutional AnimalCare and Use Committee of Texas A&M Health Science Center College ofMedicine.

Animal Models of Injury and Treatment

Injury was created by applying 100% ethanol to the whole corneaincluding the limbus for 30 seconds followed by rinsing with 10 ml ofbalanced salt solution. Then, the whole corneal and limbal epitheliumwas scraped mechanically using a surgical blade.

For injection of the recombinant human SN (PolyPeptide Laboratories,Hillerød, Denmark) or HSPB4 (Enzo Life Sciences, Plymouth Meeting, Pa.),2 μl of the proteins in PBS (0.2 ng SN in 2 μl PBS or 100 ng HSPB4 in 2μl PBS; the amount of SN or HSPB4 detected in the cornea at 15 min afterinjury) were injected using a 32 gauge needle into the corneal stromanear the temporal limbus. The proteins were purified by endotoxinbinding columns and sterilized prior to use according to themanufacturer's instructions (EndoClear, blue; Hycult Biotech Inc.Plymouth Meeting, Pa.), and tested to be free of detectable levels ofGram-negative bacterial endotoxins (<0.01 EU/ml) or proteins (1 ng/ml)using the Limulus amoebocyte lysate kit (Hycult Biotech Inc.) and E.coli HCP ELISA kit (Cygnus Technologies, Southport, N.C.).

For macrophage depletion, either 100 μl of clodronate-encapsulatedliposome (5 mg clodronate per ml suspension; Encapsula Nano Sciences,Nashville, Tenn.) or the same volume of PBS-encapsulated liposomes wereinjected subconjunctivally near the limbus on day −2 (2 days beforeinjury) and on day 0 (day of injury). The injection was dispensed overfour quadrants (25 μl each) so that a circular bleb around the corneawas formed.

For blocking the release of SN from nerve endings, 20 μl of 2 mMdiltiazem solution in isotonic saline (Sigma-Aldrich, St. Louis, Mo.)was applied topically to the cornea 15 min prior to injury (Gonzalez, etal., Invest. Ophthalmol. Vis. Sci., Vol. 34, No. 12, pgs. 3329-3335(1993). For blocking HSPB4 in the cornea, either mouse monoclonal orrabbit polyclonal antibodies to rat HSPB4 (10 μg or 50 μg in 100 μl PBS;Abcam, Cambridge, Mass.) were injected subconjunctivally near the limbusright before the injury. The same concentration of isotype lgG also wasinjected as control.

To evaluate the effect of TLR2 inhibition in the injured cornea, rhTSG-6(2 μg in 5 μL of PBS; R&D Systems, Minneapolis, Minn.) or the samevolume of PBS was injected into the anterior chamber of the rat eyeimmediately after injury.

Cells and Cell Lines

Murine macrophages (RAW 264.7) were obtained from ATCC (Rockville, Md.).Human embryonic kidney (HEK) 293 cells transfected with vectorsexpressing human TLR2 or TLR4 plus a vector expressing an alkalinephosphatase reporter gene under the control of an inducible NF-kBpromoter were purchased from InvivoGen (HEK-BIue™-hTLR2 andHEK-Blue™-hTLR4; San Diego, Calif.). A control cell line not expressingeither TLR2 or TLR4 also was obtained and used (HEK-Blue™-Nul11). Thestable cell line expressing human CD44 (Origene, Rockville, Md.) orPCDNA 3.1 control vector (Invitrogen) was generated. The primary humankeratocytes were obtained from ScienCell (Carlsbad, Calif.), and used atpassage 5.

Cell Injury Induction

To see the effects of injured cells in vitro, necrotic corneal tissueextracts were prepared. Rat corneas were homogenized in PBS (100 μl perone cornea) using a motor-driven homogenizer followed by fivefreeze-thaw cycles and 37° C. for 5 hours (Chen, et al., Nat. Med., Vol.13, No. 7, pgs. 851-856 (2007). After centrifugation at 12000 rpm for 5min, the supernatants were prepared as necrotic extracts. Some ofnecrotic extracts were heat-treated (100° C., 20 min). Necrotic extractswere incubated with the cells in culture at a 1:10 dilution for 2 hours.To evaluate the effect of HSPB4, either antibodies to HSPB4 (10 μg or 50μg) or isotype lgG antibodies also were added to the cultures.

To evaluate the effects of sHSPs and SN, either crystallins (HSPB4,HSPB5, βB crystallin; 0.001 to 10 μg/ml) or SN (0.1 to 10 ng/ml) wereadded to cultures and the cultures were incubated for 2 hours. To seethe effect of TSG-6, 100 ng/ml or 500 ng/ml of rhTSG-6 were added to thecultures. To rule out the possibility of bacterial or LPS contaminationof sHSPs, all in vitro experiments were done in the presence ofpolymyxin B (10 μg/ml) for neutralization of LPS. Moreover, foradditional control experiments. sHSPs denatured by heat (100° C., 20min) were used in parallel sets of experiments. To see whetherkeratocytes express sHSPs in response to injury, either necroticextracts or H₂O₂ (100 to 500 nM) were added to the cultures ofkeratocytes.

Measurement of the Myeloperoxidase Amount in the Cornea

For a quantitative measure of neutrophil infiltration, the corneas wereassayed for the myeloperoxidase (MPO) concentration (Rat MPO ELISA kit;HyCult biotech) as reported previously (Oh, et al., Proc. Nat. Acad.Sci., Vol 101, No. 39, pgs. 16875-16880 (2010). For protein extraction,the cornea was cut into small pieces and lysed in 150 μl of tissueextraction reagent (Invitrogen, Carlsbad, Calif.) containing proteaseinhibitor cocktail (Roche, Indianapolis, Ind.). The samples weresonicated on ice using an ultrasound sonicator. After centrifugation at12,000 rpm at 4° C. for 20 min, the cleared supernatant was collectedand assayed for levels of MPO.

Microarrays

RNA target for microarrays was prepared using the 3′ IVT Express Kit(Affymetrix) according to manufacturer's instructions. Briefly, 200 ngof total RNA was used to synthesize first strand cDNA. The cDNA then wasconverted into double-stranded cDNA and used in in vitro transcriptionto synthesize biotinylated cRNA. The cRNA was purified with magneticbeads, fragmented, and 12.5 μg were used in the hybridization ontoRG-230 2.0 arrays. The arrays were stained, washed, and scanned forfluorescence. Microarray data was normalized and analyzed using thePartek Genomics Suite 6.4 (Partek) and dChip software. For comparativeanalysis, data were filtered based on fold changes of 2 or more (eitherup- or down-regulated). For the hierarchical clustering analysis datawere filtered using a coefficient of variation higher than 0.6 and apresence call of at least 33%. The expression levels of the filteredgenes were standardized and used in hierarchical clustering. A total 6clusters were selected in each hierarchical clustering on the similarlevel of hierarchy and studied for enriched Gene Ontology tags based onhypergeometric distribution.

Real-Time RT-PCR

Total RNA from the cornea or the cells was extracted (RNeasy Mini kit;Qiagen, Valencia, Calif.) and used to synthesize double-stranded cDNA byreverse transcription (SuperScript III; Invitrogen). Real-timeamplification was performed (Taqman Universal PCR Master Mix AppliedBiosystems, Carlsbad, Calif.) and analyzed on an automated instrument(7900HT Fast Real-Time PCR System; Applied Biosystems). PCR probe setswere purchased commercially (Taqman Gene Expression Assay Kits, AppliedBiosystems). For assays, reactions were incubated at 50° C. for 2 min,95° C. for 10 min, and then 40 cycles at 95° C. for 15 sec followed by60° C. for 1 min. For normalization of gene expression, 18S rRNA probe(Taqman Gene Expression Assays ID, Hs03003631_g1) was used as internalcontrol. The threshold cycle (Ct) was used to detect the increase in thesignal associated with an exponential growth of PCR products during thelog-linear phase. The expression of molecules was calculated using thealgorithm 2^(−ΔΔCt).

Western Blot

Clear lysates prepared as described above were measured for proteinconcentration, and a total of 10 μg protein was fractionated by SDS-PAGEon 10% bis-tris gel (Invitrogen), transferred to nitrocellulose membrane(Invitrogen), and blotted with antibodies against SN (PhoenixPharmaceuticals, Burlingame, Calif.) or HSPB4 (Abeam).

ELISAs

Protein was extracted from the cornea as described above, and wasassayed for levels of pro-inflammatory cytokines and chemokines withcommercial ELISA kits for IL-6, IL-0, and CXCL1/CINC-1 (Quantikine kit;R&D Systems), and for CCL2/MCP-1 (Immunoassay Kit; Invitrogen). ForHSPB4 measurement, mouse monoclonal anti-rat antibody to HSPB4 (Abeam)was used as a capture antibody (4 μg/ml) and rabbit polyclonal anti-ratantibody to HSPB4 (Abeam) as a secondary antibody (400 ng/ml).

Release of HSPB4 in Injured Cornea

To measure the amount of HSPB4 released from the cornea after injury,the corneas of rats were harvested immediately after the injury andcultured at 37° C. with 5% CO₂ for 12 hours. Every two hours, theculture medium was changed and the concentration of HSPB4 in conditionedmedium during each time frame was measured by ELISA.

Histopathology

The cornea was excised after the rat was sacrificed and fixed in 10%paraformaldehyde. The cornea was cut into 4 μm sections and stained withthe hematoxylin-eosin (H&E) or subjected to immunohistochemistry. Theformalin-fixed corneal section was deparaffinized with ethanol andantigen was retrieved using an epitope retrieval solution (IHC WORLD,Woodstock, Md.). The rabbit polyclonal anti-rat antibody to neutrophilelastase (1:200, Abcam), the mouse monoclonal anti-rat antibody tosecretogranin II (1:200, Abcam), or the mouse monoclonal anti-ratantibody to HSPB4 (1:200, Abcam) were used as primary antibodies, andthe anti-rabbit IgG (1:5000, Abcam) or the anti-mouse IgG (1:5000,Abcam) as secondary antibodies. The DAPI solution (VECTASHIELD MountingMedium; Burlingame, Calif.) was used as counterstaining.

Aconitase Activity Assay

To evaluate the oxidative damage in the cornea by injury (Ma, et al.,Biochem, Biophys. Acta, Vol. 1790, No. 10. pgs. 1021-1029 (2009), lossof aconitase activity in the corneal lysates was measured using anaconitase assay kit according to the manufacturer's protocol (CaymanChemical Company, Ann Arbor, Mich.)

NF-kB Translocation Assays

About 1×10⁵ mouse macrophages were plated in 8 well chamber slides(Lab-Tek II Chamber Slide; Nalge Nunc, Rochester, N.Y.) and incubatedfor 1 hour in 0.2 mL of 2% FBS in α-MEM with or without 10 μg/mL HSPB4.The cells were washed twice with PBS by centrifugation and were fixedwith 100% methanol for 5 min. The cells were washed with PBS and blockedwith Image-iT™ FX Signal Enhancer (Invitrogen) The cells then wereincubated with 1 μg/mL of anti NF-kB p65 antibody (Abcam) in blockingbuffer (5% BSA in PBS) overnight at 4° C. The samples were incubated for1 hour with a 1:2000 dilution of the secondary antibody of anti-rabbitIgG (Alexa Fluor® 488 goat; Invitrogen). The DAPI solution was used tostain the cell nuclei. The slides were visualized with fluorescentmicroscopy using an upright microscope (Eclipse 80i, Nikon, Melville,N.Y.)

Statistical Analysis

Comparisons of parameters among the groups were made by the Student's ttest, non-parametric Mann-Whitney test or Pearson's correlation testusing SPSS software (SPSS 12.0). Differences were considered significantat p<0.05.

Results

Two Phases of Neutrophil Infiltration after Sterile Injury to the Cornea

The corneas of Lewis rats, were injured by exposing them to 100% ethanolfor 30 seconds and scraping of the cornea and limbus to remove both theepithehum and stem cells found in the limbus. As described previously(Oh, et. al, Proc. Nat. Acad. Sci., Vol. 107, No. 34, pgs. 16875-16880(2010), neutrophil infiltration was monitored by assays formyeloperoxidase (MPO) that is stored within neutrophil granules andreleased by activation of the cells (Borregard, et al., Blood, Vol. 89,pgs. 3503-3521 (1997). The neutrophil infiltration occurred in the twophases. There was a small initial phase that began within about 15 min,and reached a plateau level at 4 to 8 hours (Phase I in FIG. 31A). Afterthe plateau, a much larger infiltration of neutrophils followed andreached a maximum at 24 to 48 hours (Phase II in FIG. 31A). Theneutrophils then gradually disappeared in a recovery phase over 48 hoursto 7 days.

Search for Candidate Signals for Phase I and Phase II

As a strategy to identify candidate signals that initiated the twophases, microarrays were used to survey the response of the cornea toinjury. Based on the temporal pattern of gene expression, the genes wereclassified into three groups (FIG. 31B, FIG. 31D). About 842 Group Agenes were up-regulated, about 108 Group B genes were up-regulated, andabout 307 Group C genes were up-regulated (FIG. 31D). Focus was directedto the Group A genes, because they were expressed earlier and thereforemore likely to include stimuli for Phase I and II (FIGS. 31 B and C).Most of the Group B genes were the molecules related to apoptosis/deathand defense response (See Table 11 below, FIG. 31D). Most of the Group Cmolecules were pro-inflammatory chemokines and cytokines (See Table 11below, FIG. 31D, FIG. 32M), and therefore were likely to be the genesthat peaked late in the inflammatory responses. Most of the Group Agenes were genes for nerve/neurotransmission-related and structuralproteins of the eye (See Table 11 below.) From the category of Group Agenes, the following were selected as attractive candidates forinflammatory signals: The neuropeptide secretoneurin (SN) because it wasshown previously to activate chemotaxic migration and transendothelialextravasation of blood cells (Helle, Regul Pept., Vol. 165, No. 1, pgs.45-51 (2010); Taupenot, et al., N. Engl. J. Med., Vol. 348, No. 12, pgs.1134-1149 (2003), and two small heat shock proteins (HSPB4 and HSPB5)because some heat shock proteins were previously shown to act as DAMPs(Joly, et al., J. Innate Immun., Vol. 2, No. 3, pgs. 238-247 (2010); VanWijk, et al., J. Leukoc. Biol., Vol. 88, No. 3, pgs. 431-434 (2010);Quintana, et al., J. Immunol., Vol. 175, No. 5, pgs. 2777-2782 (2005);Asea, et al., Nat. Med., Vol. 6, No. 4, pgs. 435-442 (2000).

TABLE 11 Microarray analysis of gene expression profiles in the corneaat 4 hours and 24 hours after injury. The top 20 transcripts upregulatedby injury were shown in each group (Group A. B, and C). Change (x-Fold)Injured (4 h)/ Injured (24 h)/ Gene Title Gene symbol Probe set con conGroup A crystallin, alpha A Cryaa 1370279_at 19.468 1.620 crystallin,beta B1 Crybb1 1369985_at 18.541 1.062 crystallin, gamma C Crygc1370292_a_at 17.911 1.112 secretogranin II ScgII 1368044_at 15.847 1.033claudin 2 Cldn2 1375933_at 15.246 1.033 crystallin, beta A1 Cryba11371408_at 14.628 −3.593 crystallin, gamma B Crygb 1371413_x_at 14.3111.025 crystallin, gamma D Crygd 136770._at 13.957 1.054synaptosomal-associated Snap25 1387073_t 13.199 1.580 protein 25Galectin-related inter-fiber Grifin 1386936_at 12.710 −1.129 proteinsolute carrier family 6 Slc6a1 1368170_at 12.359 −1.704(neurotransmitter transporter, GABA), member 1 crystallin, gamma S Crygs1388435_at 11.969 −1.096 Calbindin Calb1 1370201_at 11.542 −1.184crystallin, beta A2 Cryba2 1388385_at 10.349 −1.497 crystallin, beta B2Crybb2 1367684_at 9.918 −1.738 retinol binding protein 3, Rbp31376777_at 9.789 −1.434 interstitial collagen, type II, alpha 1 Col2a11387767_a_at 8.615 −1.224 phosphodiesterase 6A, Pde6a 1393426_at 8.500−1.011 cGMP-specific, rod, alpha complexin 3 Cplx3 1384779_at 8.061−1.099 crystallin, alpha B Cryab 1370026_at 7.197 1.812 crystallin, betaA4 Cryba4 1367608_at 7.168 −1.749 Group B interleukin 6 Il6 1369191_at340.974 293.81 colony stimulating factor 3 Csf3 1369529_at 70.050 62.614(granulocyte) matrix metallopeptidase 13 Mmp13 1388204_at 30.118 28.285prostaglandin E synthase Ptges 1368014_at 19.449 15.069 metallothionein2A Mt2A 1388271_at 17.356 13.067 superoxide dismutase 2, Sod2 1370173_at16.915 11.803 mitochondrial interleukin I alpha Illa 1371170_a_at 14.6375.275 prostaglandin-endoperoxide Ptgs2 1368527_at 10.861 8.108 synthase2 metallothionein 1a Mtla 1371237_a_at 9.483 6.815 lipocalin2 Lcn21387011_at 9.013 9.387 immediate early response 3 Ier3 1388587_at 5.5525.781 prostaglandin E synthase Ptges 1368015_at 5.433 5.051 sixtransmembrane Steapl 1393706_at 5.221 4.467 epithelial antigen of theprostate 1 cAMP responsive element Crem 1393550_at 5.213 4.998 modulatortransferrin receptor Tfrc 1388750_at 5.182 5.061 mitogen-activatedprotein Map3k8 1369393_at 4.935 5.302 kinase 8 B-cell translocation gene2, Btg2 1386994_at 4.931 4.677 anti-proliferative runt relatedtranscription Runx 1 1368914_at 4.924 3.913 factor 1 similar to F-boxonly RGD1563982 1375041_at 4.717 3.358 protein 27 growth arrest, DNA-Gadd45a 1368947_at 4.026 3.756 damage-inducible, alpha Group C secretoryleukocyte peptide Slpi 1367998_at 54.415 533.847 chemokine (C—X—C motif)Cxcl2 1368760_at 52.728 404.344 ligand 2 S100 calcium binding S100a91387125_at 56.114 207.701 protein A9 interleukin 1 beta Illb 1398256_at33.522 198.673 chemokine (C-C motif) Ccl7 1379935_at 40.595 159.69ligand 7 S100 calcium binding S100a8 1368494_at 33.206 155.69 protein A8chemokine (C—X—C motif) Cxcl3 1370633_at 7.895 128.417 ligand 3chemokine (C-C motif) Cc13 1369815_at 6.493 95.538 ligand 3 interleukin1 receptor, type Illr2 1387180_at 24.159 60.257 II chemokine (C—X—Cmotif) Cxcl3 1370634_x_at 4.934 59.166 ligand 3 interleukin I alpha Illa1368592_at 2.953 54.174 Fc fragment of IgG, low Fcgr2a 1367850_at 8.70841.195 affinity Ila. receptor Cd53 molecule Cd53 1368518_at 6.748 31.988chemokine (C-C motif) Ccr1 1370083_at 6.359 28.512 receptor I chemokine(C—X—C motif) Cxcl3 1388032_a_at 2.331 24.419 ligand 3 colonystimulating factor 3 Cst3r 1386009_at 8.094 21.987 receptor Fc fragmentof IgG, high FcgrIa 1393038_at 3.611 21.032 affinity Ia, receptorlaminin, gamma 2 Lamc2 1379340_at 5.817 20.577 immunoglobulin Igsf61387687_at 2.915 20.148 superfamily, member 6 complement component 3 C31368000_at 6.292 15.602 Symbols: Injured (4 h)/con, cornea at 4 hoursafter injury vs. cornea right after injury; Injured (24 h)/con, corneaat 24 hours after injury vs. cornea right after injury; ‘−’ meansdownregulation. The values are the results from collective samples of n= 4 per each group.

SN as a Candidate for Phase I and HSPB4 for Phase II

Data on the time course of expression were consistent with SN serving asan initiating signal for Phase I. SN was not detected in extracts ofuninjured cornea, but appeared both in corneal extracts and the serum ofthe rats within 0.25 hour of the injury (FIGS. 32A and B). The levels ofSN in extracts of injured corneas and the serum decreased at 0.5 hourand then increased apparently as a result of increased expression of thegene (FIGS. 32A-D). In contrast, there were little changes in theexpression of genes for substance P and calcitonin gene-related peptide(CGRP), two other neuropeptides known to be expressed in the cornea(FIG. 32D) (Troger, et al., Brain Res. Rev., Vol. 53, No. 1, pgs. 39-62(2007).

Similar data on the time course of expression were consistent with HSPB4serving as a stimulus for Phase II. Uninjured cornea contained lowlevels of HSPB4 protein but the amount increased beginning after 0.5hour of the injury and reached a peak at about 4 hours (FIGS. 32E andF). A similar time course was observed in the release of HSPB4 into themedium in experiments in which corneas were injured in vivo and thenincubated ex vivo (FIG. 32G). As expected from the microarray data,there was increased expression of mRNAs for HSPB4 and related genes fromGroup A (FIG. 32I); however, the increases in expression of HSPB4 weredelayed compared to the increase in the mRNA for SN (compare FIGS. 32Dand I). Immunohistochemistry of injured cornea was consistent with theresults. The SN-immunoreactive nerves were increased in the cornea at 2hours following injury (FIG. 32C). The expression of HSPB4 was increasedat about 4 hours following injury (FIG. 32H).

In addition, the expressions of SN and HSPB4 were dependent on theseverity of injury. The concentrations of both the proteins and mRNAswere higher in the cornea or serum following severe injury (30 secethanol and scraping) compared to mild injury (15 sec ethanol andscraping) (FIG. 33F).

Keratocytes from the Corneal Stroma Synthesized HSBP4 in Response toInjury

To define the cellular origin of HSPB4 in the injured cornea,keratocytes that are fibroblast-like cells from the corneal stroma wereincubated with extracts of the cornea that were made necrotic byrepeated freezing and thawing. The necrotic extracts induced theexpression of HSPB4 in the keratocytes (FIG. 32J). The necrotic extractsdid not increase significantly the expression of a second small heatshock protein HSPB5 or a third Group A gene, βB crystallin. Most sterileinflammations produce increases in reactive-oxygen species (ROS)(Kolnitzer, et al., Ann. N.Y. Acad. Sci., Vol. 1203, pgs 45-52 (2010);Martinon, Eur. J. Immunol., Vol. 40, No. 3, pgs. 616-619 (2010). Assaysof injured cornea demonstrated a rapid decrease in aconitase activity, areflection of an increase in ROS (FIG. 32K). As expected, incubation ofkeratocytes with H₂O₂ to increase ROS also produced increased expressionof HSPB4 (FIG. 32L).

Recombinant SN Reproduced Phase I and Recombinant HSPB4 Reproduced PhaseII

Injection of recombinant SN into the stroma of the cornea stimulated theneutrophil infiltration of Phase C (FIG. 33A). The effect was negatedpartially by topical application of a calcium blocker (Diltiazem) (FIG.33D) that inhibits release of neuropeptides (Gonzalez, et al, Invest.Ophthalmol. Vis. Sci., Vol. 34, No. 12, pgs. 3329-3335 (1993). Theresults therefore indicated that SN served as a major stimulus for PhaseI, but they did not exclude the possibility that it acted in concertwith other signals released by the injured cornea.

Injection of recombinant HSPB4 that was pyrogen-free (see Methods)stimulated the neutrophil infiltration of both Phase I and Phase II(FIGS. 33B and C). Reproduction of Phase I was explained apparently bythe protein being injected earlier than it appears in the tissue afterinjury to the cornea (FIGS. 32E-H). Injection of recombinant HSPB4 intoone region of the cornea also reproduced the opacity produced by sterileinflammation (FIG. 33C). The role of HSPB4 was confirmed by experimentsin which antibodies to the protein were injected into the corneal stromaimmediately after the injury (FIG. 33E). The antibodies to HSPB4inhibited significantly the Phase II inflammatory response in the corneaafter the injury. Also, the neutrophil infiltration of Phase II in thecornea after injury was decreased significantly in the corneas of HSPB4knockout mice, compared to wild-type controls (FIG. 33F).

HSPB4 Activated Resident Macrophages Through TLR2/NF-kB Signaling.

To identify the cells that responded to HSPB4 in the cornea, residentmacrophages were depleted from the corneas of Lewis rats by injectingclodronate liposomes subconjunctivally, and then recombinant HSPB4 wasinjected into the corneal stromas. Macrophage depletion was confirmedwith immunostaining for macrophage-specific markers CD68 and CD11b (FIG.33G). The protein did not reproduce Phase II in rats in whichmacrophages were depleted (FIG. 34A), suggesting that the effects ofHSPB4 were dependent on the presence of resident macrophages. Similarly,the inflammation was decreased markedly in the cornea afterchemical/mechanical injury when resident macrophages were depleted priorto an injury (FIG. 34L), indicating a crucial role of residentmacrophages in sterile injury-induced inflammation of the cornea. Inparallel experiments, it was observed that neutrophil infiltration ofPhase II, but not Phase I, was suppressed significantly by anintraocular injection of the anti-inflammatory protein, TSG-6 (FIG.34B), that inhibits TLR2/NF-kB signaling in macrophages (Choi, Blood, inpress; Lesley, et al., J. Biol. Chem., Vol. 279, pgs. 25745-25754(2004)). Also, TSG-6 suppressed significantly the Phase II inflammatoryresponse caused by HSPB4 injection to the corneal stroma (FIG. 34C). Theresults suggested therefore that HSPB4 signaled Phase II by activatingTLR2/NF-kB signaling in resident macrophages.

To test the effects of HSPB4 on macrophages, macrophages were incubatedwith extracts of necrotic corneas. The extracts increased the expressionof the pro-inflammatory cytokines IL-1α, IL-1β, and IL-6 (FIG. 34D);however, heat inactivation of the extracts inhibited their effect,suggesting that the active factor(s) in the extracts was (were)protein(s). Addition of either a polyclonal or monoclonal antibody toHSPB4 suppressed significantly the effect of the necrotic extracts onmacrophage activation, indicating that one of the active factors innecrotic corneal extracts was HSPB4 (FIG. 34D). In addition, recombinantHSPB4 increased the expression of the pro-inflammatory cytokines bymacrophages in a dose-dependent manner (FIG. 34E). As expected, HSPB4caused translocation to the nucleus of the NF-kB complex in macrophages(FIG. 34F). To test whether HSPB4 signaled through the TLR2/NF-kBpathway, a reporter cell line transduced was used to assay TLR2/NF-kBsignaling (HEK-TLR2). Experiments in a HEK-TLR2 cell line demonstratedthat the necrotic extracts of the cornea increased TLR2/NF-kB signalingand that antibodies to HSPB4 inhibited the effect (FIG. 34G).Recombinant HSPB4 stimulated NF-kB signaling in the same cell line in adose-dependent manner (FIG. 34H). HSPB4 acted primarily through TLR2; ithad a smaller effect in the reporter cell line that expressed TLR4 andno effect in the reporter cell without either receptor (FIGS. 34I andJ); however, HSPB4 did not stimulate keratocytes in culture to expresspro-inflammatory cytokines (FIG. 35F). Also, other Group A moleculessuch as HSPB5 and βB-crystallin had no effect on pro-inflammatorycytokine production in macrophages or on NF-KB activation in HEK-TLR2cells (FIG. 34K). In addition, SN did not induce the expression ofpro-inflammatory cytokines in either macrophages or keratocytes in vitro(FIG. 35F). Similarly, HSPB4 did not stimulate keratinocytes in cultureto express pro-inflammatory cytokines. (FIG. 35F). Together, the resultsindicated that HSPB4 was a principal DAMP for Phase II and acted throughthe activation of resident macrophages in the cornea.

TSG-6 Suppressed HSPB4-Induced Activation of Macrophages.

TSG-6 was shown previously to provide a potential therapy for chemicalinjuries of the cornea but its mechanism of action was not established(Oh, Proc. Nat. Acad. Sci., 2010). Therefore, the hypothesis that TSG-6inhibited the Phase II response, but not Phase I (FIGS. 34B and C) wastested by decreasing the HSPB4 induced activation of macrophages.Recombinant TSG-6 decreased expression of pro-inflammatory cytokines inmacrophages stimulated by HSPB4 (FIGS. 35A and B). Because the proteinwas shown previously to inhibit TLR2/NFkB signaling in macrophages byinteraction with CD44 (Choi, Blood, in press; Lesley J, Gal I, Mahoney DJ, et al., TSG-6 modulates the interaction between hyaluronan and cellsurface CD44. J Biol Chem., 2004; 279:25745-25754). Whether the effectsof TSG-6 were CD44-dependent also were examined. As expected, TSG-6 hadno significant effect on NF-kB signaling in the HEK-TLR2 reported cellline unless the cell line were transduced to express CD44 (FIGS. 35C andD); however, TSG-6 had no effect on the parent cell line that did notexpress CD44. Also, TSG-6 had no effect the Phase II inflammatoryresponse after chemical/mechanical injury to the cornea of CD44 knockoutmice (FIG. 35E), indicating that the action of TSG-6 an macrophages wasCD44-dependent.

Discussion

As summarized in FIG. 36, the results here demonstrated that there weretwo distinct phases of neutrophil infiltration in the cornea aftersterile injury: (a) a small initial Phase I that began within 15 min,and reached a plateau level in 4 to 8 hours, and (b) a much larger PhaseII with a peak at 24 to 48 hours. In search for the stimuli for Phase Iand II responses, the upregulated molecules after an injury werescreened on the assumption that the molecules tissues actively produceafter injury may be the major stimuli to induce sterile inflammation.

A major stimulus for the Phase I response was the neuropeptide SN. ThePhase I response began within 15 min of the injury and, as indicated bythe depletion experiments with clodronate, it did not requireparticipation of macrophages. Neuropeptides are attractive candidatesfor Phase I stimuli because they are preformed and released rapidly fromsensory nerve endings in response to injury. Also, the cornea is one ofthe most densely innervated tissues. Intrastromal injection ofrecombinant SN reproduced the Phase I response and an inhibitor ofneuropeptide blocked the effect. As indicated by experiments withmacrophage depletion, Phase I did not require participation of residentmacrophages. SN did not elicit the further, intense infiltration ofneutrophils in Phase II that was mediated by activation of residentmacrophages. Therefore, the Phase I response was independent of thePhase II response.

Neuropeptides are attractive candidates for the stimuli of the initialinflammatory responses of the tissues because they are preformed and arereleased rapidly from nerve endings by injury. Neuropeptides may beimportant particularly in the response of the cornea to an injurybecause the cornea is one of the most heavily innervated tissues in thebody. (Nishida, Cornea, 2^(nd) Edition (Krachmer, et al., eds.), pg. 4.(Elsevier Mosby, Philadelphia, 2005)). SN is a member of the graninfamily of neuropeptide/neurotransmitter, and it contributes to innateimmunity by activating chemotactic migration and transendothelialextravasation of immune cells (Helle, 2010; Taupenot, 2003).Importantly, SN was shown to be as effective as TNF-α in stimulatingtransmigration of neutrophils through an endothelial cell barrier(Kahler, et al., Exp. Lung. Res., Vol. 27, No. 1, pgs. 25-46 (2001);Kahler, et al., Regul. Pept., Vol. 105, No. 1, pgs. 35-46 (2002). Also,recent in vivo studies suggest that SN has a role not only in neurogenicinflammation, but also a more complex role in common diseases, such asmyocardial infarction, chronic heart failure, essential hypertension,rheumatoid diseases, or acute inflammatory bowel syndromes thanappreciated previously (Helle, 2010; Taupenot, 2003; Wiedermann,Peptides, Vol. 21, No. 8, pgs. 1289-1298 (2000).

The data demonstrated that the principal DAMP for the Phase II responsewas the small heat shock protein HSPB4. The recombinant HSPB4 proteinfree of pyrogens reproduced the Phase II response by activating residentmacrophages via the TLR2 NF-KB signaling pathway. The role of HSPB4 wasconfirmed by the observation that the Phase II response in injuredcornea was diminished greatly by injection of blocking antibodies toHSPB4. Injury to the cornea released HSPB4 extracellularly. Addition ofnecrotic extracts from corneal tissues increased the synthesis of HSPB4by stromal keratocytes, probably as a result of increased ROS and otherstimuli in the necrotic cornea. Moreover, HSPB4 stimulated theTLR2/NF-KB signaling in macrophages to release a cascade ofpro-inflammatory cytokines such as IL-1α and IL-1β.

The role of HSPB4 as a DAMP is consistent with previous observationswith the protein and other heat shock proteins such as HSP60, 70, 72, or96. (Lehnardt, et al. J. Neurosci; Vol. 28, pgs. 2320-2331 (2008); Asea,et al., Nat. Med., Vol. 6, pgs. 435-442 (2000); Wheeler, et al.; Respir.Res., Vol. 10, pgs. 31-43 (2009). Huang, et al., J. Immunol., Vol. 182,pgs 4965-4973 (2009); Chen, et al., Eur. J. Immunol., Vol. 40, pgs.1541-1544 (2010)). HSPB4 is a member of small molecular weight heatshock proteins (sHSPs) that range in size from 15-30 kDa. Like otherheat shock proteins, sHSPs are induced by heat and other stimuli such asoxidative and inflammatory stresses, and they act as a chaperones toprotect cells from damage produced by misfolded proteins. However, HSPshave paradoxical effects. (Kobba, et al., Shock, Dec. 9, 2010 onlinepublication; DeMeester, et al., FASEB J., Vol. 15, pgs. 270-274 (2000);Chen, et al., Inflamm. Allergy Drug Targets, Vol. 6, pgs. 91-100(2007)). Heat shock pre-treatment induces intracellular HSPs to protectagainst inflammation (Ousman, et al., Nature, Vol. 448, pgs. 474-479(2007); Saraswathy, et al., Invest. Ophtalmol. Vis. Sci., Vol. 51, No.7, pgs. 3680-3686 (2010); Munemasa, et al., Invest. Ophthalmol. Vis.Sci., Vol. 50, No. 8, pgs. 3869-3875 (2009), whereas after inflammatoryinjury has been primed or cell integrity has been compromised, heatshock induces expression of HSPs that are released from damaged orstressed cells and exacerbates cell injury (Asea, et al., Nat. Med.,Vol. 6, No. 4, pgs. 435-442 (2000); Quintana, et al. J. Immunol., Vol.175, No. 5, pgs. 2777-2782 (2005); Lehnardt, et al., J. Neurosci, Vol.28, No. 10, pgs. 2320-2331 (2008); Huang, et al., J. Immunol., Vol. 182,No. 8, pgs. 4965-4973 (2009); Wheeler, et al., Respir. Res., Vol. 10,pg. 31 (2009). Furthermore, HSPs are known to function as an endogenousadjuvant to facilitate the binding of other danger signals to immunecells and thus to enhance the immunogenicity. (Chen, et al., Eur. J.Immunol., Vol. 40, pgs. 1541-1544 (2010)). Consistent with thesereports, it now is reported that a small HSP, HSPB4, stimulatedmacrophages in vitro through the TLR2/NF-kB pathway and acted as theprincipal DAMP for the Phase II inflammatory response in the injuredcornea. On the other hand, another small HSP, HSPB5, did not induce thisresponse.

The identification of HSPB4 as the principal DAMP for the Phase IIinflammatory response has several implications for potential therapies.One is that the results provide an explanation for the beneficialeffects observed previously with applications of TSG-6 followingchemical injury to the cornea (Oh, 2010). TSG-6 interacts with CD44 onmacrophages and decreases thereby the HSPB4 induced TLR2/NFkB signalingthat has been induced by HSPB4 released from injured cornea. (Choi, inpress).

Another implication is that antibodies or antagonists to HSPB4 mightserve as a therapy alone or in combination with TSG-6 to modulate orprevent sterile inflammation of the cornea. (Oh, 2010). Still anotherimplication is that the results may provide a guide to developingtherapies for other diseases of the eye or of nonocular tissues. HSPB4was shown to be expressed and upregulated in the retina in models foruveitis (Sarawathy, et al., Invest. Ophthalmal. Vis-Sci., Vol. 51, pgs.3680-3686 (2010); Rao, et al., Invest. Ophthalmol. Vis. Sci., Vol. 49,pgs. 1161-1171 (2008)), glaucoma (Munemasa, et al., Invest. Ophthalmol.Vis. Sci., Vol. 50, pgs. 3869-3875 (2009)), age-related maculardegeneration (Kapphahn, et al., Biochemistry, Vol. 42, pgs. 15310-15325(2003)), and hypoxic retinopathy (Miyara, et al., Jpn. J. Ophthalmol.,Vol. 52, pgs. 84-90 (2008)). HSPB4 also is known to be expressed innonocular tissues such as spleen, thymus, pancreas, and kidney. (Deng,et al., Biochem. Biophys. Acta, Vol. 1802, pgs. 621-631 (2010);Srinivasan, et al., J. Biol. Chem., Vol. 267, pgs. 23337-23341 (1992)).Because sterile inflammation contributes to the pathogenesis of manydiseases (Chen, et al. Nat. Rev. Immunol., Vol. 10, pgs. 826-837 (2010);Rock, et al., Ann. Rev. Immunol., Vol. 28, pgs. 321-342 (2010); Spite,et al., Circ. Res., Vol. 107, pgs. 1170-1184 (2010)), the strategy ofmodulating the activities of HSPs or related receptors using TSG-6 orantibodies or antagonists to extracellular HSPs may present useful drugtargets to treat a variety of inflammatory diseases where HSPs are theprimary signals for inflammation, such as ischemia-reperfusion injury,acute lung injury, various kidney diseases, atherosclerosis, obesity,and diabetes. (Gill, et al., Free Radic. Biol. Med., Vol. 48, pgs.1121-1132 (2010)).

The disclosures of each and every patent, patent application,publication, database accession number, and depository accession numbercited herein are hereby incorporated herein by reference in theirentirety to the same extent as if each patent, patent application,publication, database accession number, and depository accession numberwere incorporated individually by reference.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

REFERENCES

-   Aggarwal et al., 2005, Blood 105:1815-22-   Akiyama et al., 2002, Glia 39(3): 229-36-   Armstrong et al., 1998, Angiogenesis. 2(1):93-104.-   Avila et al., 2001, Cornea 20:414-20-   Block et al., 2009, Stem Cells 27(3):670-81-   Callaghan et al., 2001, Rheumatology (Oxford) 46:105-11-   Caplan, 1990, Biomaterials 11:44-6-   Caplan et al., 2005, Tissue Eng 11:1198-1211-   Caplan et al., 2006, J Cell Biochem 98(5):1076-84-   Castro-Malaspina et al., 1980, Blood 1980 56(2):289-301-   Cauchi et al., 2008, Am J Ophthalmol 146:251-9-   Cho et al, 1998, Cornea 17:68-73-   Chen et al., 2006, Immunol Cell Biol 84:413-21-   Clegg et al., 2006, Ophthalmic Epidemiol 13:263-74-   D'Amato et al., 1994, Proc Natl Acad Sci USA 91:4082-5-   Daya et al., 2005, Ophthalmology 112:470-7-   De Ban et al., 2003. J Cell Biol 160(6) 909-18-   Dooner et al., 2004, Blood Cells Mal Dis 32(1):47-51-   Eaves et lt., 2001, Ann N Y Acad Sci 938:63-70, discussion 70-1-   Espana et al., 2003, Br J Ophthalmol 87:1509-14-   Fantes et al., 1990, Arch Ophthalmol 108:665-75-   Foster C S, Letko E, Ba-Abbad R A Stevens-Johnson Syndrome    [Internet] [updated 2007 Dec. 18, cited 2008 Mar. 7] Available from    http://emedicine.medscape.com/article/1197450-overview-   Fukuda et al., 2006, Circ Res 98(8): 1002-13-   Gao et al., 2001, Cells Tissues Organs 169(1):12-20-   Gerdoni et al., 2007, Ann Neurol 61(3):219-27-   Guilak et al., 2004, Biorheology 41(3-4):389-99-   Gupta et al., 2007, J Immunol 179(3):1855-63-   Hogg et al., 1994, J Appl Physiol 77(4): 1795-800-   Homma et al., 2004, Invest Ophthalmol Vis Sci 45:4320-6-   Horwitz et al., 2002, Proc Natl Acad Sci USA 99(13):8932-7-   Horwitz et al., 1999, Nat Med 5:309-13-   Ilari et al., 2002, Ophthalmology 109:1278-84-   Iso, et a]., 2007, Biochem Biophys Res Commun 354:700-6-   Javazon et al., 2001, Stem Cells 19:219-25-   Jenkins et al., 1993, Eye 7:629-33-   Kim et al., 2006, Brain Res 1123(1):27-33-   Koc et al., 2002, Bone Marrow Transplant 30:215-22-   Krampera et al., 2007, Bone 40(2):382-90-   Kuznetsov et al., 2001, J Cell Biol 153(5):1133-40-   Le Blanc et al., 2007, J Intern Med 262(5):509-25-   Lee et al., 2006, Proc Natl Acad Sci USA 103:17438-43-   Lee et al., 2009, Cell Stem Cell 5:54-63-   Limb et al., 2008, Br Med Bull 85:47-61-   MacDonald et al., 2002, Bioessays 24(10):885-93-   Mareschi et al., 2006, Exp Hematol 34(11) 1563-72-   Melsaether C, Rosen CL Burns. Ocular [Internet] [updated 2009 Aug.    12] Available from    http://emedicine.medscape.com/article/798696-overview-   Mertzants et al., 2005, Invest Ophthalmol Vis Sci 46:46-50-   Mets et al., 1981, Mech Ageing Dev 16(1):81-9-   Mitjanovic et al., 2007, Am J Ophthalmol 143:409-15-   Milner et al., 2003, J Cell Sci 116:1863-73-   Milner et al., 2006, Biochem Soc Trans 34:446-50-   Mishra, 2008, J Cardiovasc Med (Hagerstown) 9(2):122-8-   Moss et al, 2000, Arch Ophthatmol 118:1264-8-   Moss et al., 2008, Optom Vis Sci 85:668-74-   Munoz et al., 2005, Proc Natl Acad Sci USA 102:18171-6-   Nomura et al., 2005, Neuroscience 136(1):16t-9-   Oh et al., 2008, Stem Cells 26:1047-55-   Oh et al., 2009b, Curr Eye Res 34(2):85-91-   Oh et al., 2009a, Cytokine 46(1)100-3-   Ohtaki et al., 2008, Proc Natl Acad Sci USA 105:14638-43-   Ortiz et al., 2007, Proc Natl Acad Sci USA 104:11002-7-   Owen et al., Ciba Found Symp 136:42-60-   Penolazzi et al., 2008, Cell Biol Int 32:320-5-   Pereira et al., 1998, Proc Natl Acad Sci USA 95(3):! 142-7-   Pflugfetder et al., 2008, Am J Manag Care 14:S102-S 106-   Piersma et al., 1983, Br J Haematol 54(2):285-90-   Prockop, 1985, J Clin Invest 75(3):783-7-   Prockop et al., 1995, Annu Rev Biochem 64:403-34-   Prockop, 1997, Science 276:71-4-   Prockop et al., 2003, Proc Natl Acad Sci USA 100; 119 17-23-   Prockop, 2007, Clin Pharmacol Ther 82:241-3-   Prockop, 2009, Mol Ther 17:939-46-   Rao et al., 1999, Ophthalmology 106:822-8-   Reddy et al., 2004, Cornea 23:751-61-   Ren et al., 2008, Cell Stem Cell 0.2:141-50-   Reinhard et al., 2004, Ophthalmology 111:775-82-   Ringden et al., 2006, Transplantation 81:1390-7-   Rosada et al., 2003, Calcif Tissue 72(2):135-42-   Schaumberg et al., 2003, Am J Ophthalmol 136:318-26-   Schinkothe et al, 2008, Stem Cells Dev 17:199-206-   Schrepfer et al., 2007, Transplant Proc 39(2) 573-6-   Seo et al., 2005, J Dent Res 2005 84(10):907-12-   Sharpe et al., 2007, Tissue Eng 13:123-32-   Shi et al., 2008, Clin Exp Ophthalmol 36:501-7-   Shorn et al., 2007. Surv Ophthalmol 52:483-502-   Solomon et al., 2002, Ophthalmology 109:1159-66-   Spees et al, 2006, Proc Natl Acad Sci USA 103(5):1283-8-   Tang et al., 2007, Cell Transplant 16(2)159-69-   Ti et al., 2002, Invest Ophthalmol Vis Sci 43:2584-92-   Tseng et al., 1998, Arch Ophthalmol 116:431-41-   Tsubota et al, 1999 N Engl J Med 340:1697-703-   Ueno et al., 2007, Cornea 26:1220-7-   Wakitani et al., 1995, Muscle Nerve 18(12): 1417-26-   Wisniewski et al, 2004, Cytokine Growth Factor Rev 15: 129-46-   Woodbury et al., 2000, J Neurosci Res 61(4):364-70-   Wu et al, 2008, Cell Transplant 16(10):993-1005-   Zacharek et al., 2007, J Cereb Blood Flow Metab 27:1684-91

1-38. (canceled)
 39. A method of treating a disease or disorder of theeye in a patient, comprising: administering to said patient at least oneantibody or antibody fragment which recognizes a heat shock protein, orat least one heat shock protein antagonist wherein said antibody orantibody fragment or heat shock protein antagonist is administered in anamount effective to treat said disease or disorder of the eye in saidpatient.
 40. The method of claim 39 wherein said antibody or antibodyfragment is an antibody or antibody fragment which recognizes a heatshock protein selected from the group consisting of the small heat shockprotein HSPB4, HSP90, HSP70, HSP65, and HSP
 27. 41. The method of claim40 wherein said antibody or antibody fragment is an antibody or antibodyfragment which recognizes the small heat shock protein HSPB4.
 42. Themethod of claim 39 wherein said heat shock protein antagonist is anantagonist to HSPB4, HSP90, HSP70, HSP65, or HSP
 27. 43. The method ofclaim 42 where said heat shock protein antagonist is an antagonist toHSPB4. 44-45. (canceled)